G protein-coupled receptor (GPCR) modulation by imipridones

ABSTRACT

Imipridones has been found to selectively modulate Class A G protein-coupled receptors (GPCRs), such as the D2-like subfamily of dopamine receptors, and to be useful for the treatment of conditions and disorders in need of such modulation, such as cancers, psychiatric disorders, and bacterial infections. In addition, methods of identifying whether a subject having these condition, is likely to be responsive to a treatment regimen, such as administration of an imipridone, are provided. Furthermore, methods of assessing the effectiveness of a treatment regimen, such as administration of an imipridone, monitoring, or providing a prognosis for a subject with these condition are also provided.

BACKGROUND OF THE INVENTION

Human cells have a variety of receptors on their surfaces. Gprotein-coupled receptors (“GPCR” or “GPCRs”) form one of the largestprotein families of transmembrane receptors. The human genome hasapproximately 30,000 genes, as many as 1,000 of which encode GPCRs.GPCRs have been grouped into five classes. The first class is therhodopsin receptor family or “Class A GPCR” with 670 receptor proteins.The rhodopsin receptor family can react with various ligands includingamines (alpha group), peptides (beta group), lipid-like substances(gamma group), nucleotides, and glycoproteins (delta group), andcomprises a lot of drug target receptors. The second class is thesecretin receptor family, and has binding domains for peptide hormones.Receptors in this family are associated with homeostasis and have beenarising as important targets for drug development. The third class isthe adhesion receptor family, characterized by a GPCR proteolytic site(GPS). Development of drugs targeting this family of GPCRs has not yettaken place because they exhibit various N-terminal moieties and littleis known about their ligands. The fourth class is the glutamate receptorfamily with 22 GPCR members have so far been identified. Relativelylittle is known about the specificity of each protein. The last class isthe Frizzled/Taste2 family that encompasses 10 Frizzled receptors forwhich Wnt glycoproteins serve as ligands, 5 SMO (smoothened) receptorswhich need no ligands, and 25 Taste2 receptors which are required forsensing various tastes. Receptors including GPCRs are also classified onthe basis of the identification of endogenous ligands. Receptors bindwith known endogenous compounds or are classified as orphan receptorswhose endogenous ligands have not yet been identified.

GPCRs are found in a broad range of tissue and cell types and associatedwith many different physiological mechanisms. They are activated by awide range of ligands, e.g., hormones such as thyroid-stimulatinghormone (TSH), adrenocorticotropic hormone, glucagon and vasopressin,amines such as 5-HT, acetylcholine (muscarinic AchR), and histamines,lipids such as LPA and S1P, and signal transmitters such as amino acids,Ca²⁺, nucleic acids, peptides and light. The wide distribution anddiversity of roles that GPCRs play is evidence of their importance invarious pathological diseases. Indeed, GPCRs are involved in variousdiseases including bronchoconstriction, hypertension, diabetes,inflammation, cell death, hormone disorders, cancer, neurotransmissionand behavioral disorders. GPCRs are therefore an important area for thedevelopment of pharmaceutical products. Approximately 360 GPCRs are nowconsidered available for drug development. Of these, 46 have alreadybeen used for drug development. There are approximately an estimated 150Orphan GPCRs (oGPCRs). In the drug development field, cell membranereceptors act as selective sites for drug action and are responsible for50% of all drug targets; GPCR activity modulating drugs account for 30%of the most frequently used top 100 drugs (40 billion dollars, 9% of thetotal drug market). Therefore, GPCRs are among the most significanttargets for new drug development.

GPCRs have common structural features. They have seven hydrophobicmembrane-spanning domains, each 20-30 amino acids long, which areconnected by hydrophilic amino acid sequences of various lengths. Thereceptors have an extracellular N-terminus while the C-terminus islocated in the cytoplasm. GTP-binding proteins (G proteins) act asmediators transmitting to intracellular effectors the signals that aregenerated by binding hormones or other chemical ligands that stimulateGPCR. After ligand binding, the GPCR intracellular domain undergoes aconformational change to allow the receptor to interact with a Gprotein, which in turn activates intracellular signal transmitters suchas adenylate cyclase, phospholipase C or ion channels. This systemgenerates a signaling cascade in which many secondary transmitters actin response to the binding of one ligand to GPCR. Cells use thismechanism to detect extracellular environmental changes and to properlyreact in response to the changes. On the whole, endogenous ligandsactivate receptors with the concomitant generation of a conformationalchange, which allows association between the receptors and G proteins.Recent studies on the interaction between proteins have revealed thatGPCRs associate with various proteins such as GRK or SH2 (Src Homology2) domain-containing proteins, and adaptor Grb2 as well as G protein toparticipate in signaling transduction.

Under normal conditions, signaling transduction brings about the finalresult which is cell activation or suppression. In a physiologicalenvironment, GPCRs exist in equilibrium between their inactive andactive states in the cell membrane. Inactive receptors cannot exert abiological response in conjunction with cellular signal transductionpathways. The receptors exhibit biological responses via a signaltransduction pathway (through G proteins) only when they havestructurally changed to their active form. The receptor may bestabilized into an active form by compounds such as endogenous ligandsor drugs. Therefore, functional studies, such as cloning such genefamilies, and the identification of new ligands thereof, have the samemeaning as the development of new drug candidates, that is, siRNA,antibodies, polypeptides, effectors, inhibitors, agonists, antagonists.

Development, differentiation, homeostasis, responses to stimuli, controlof the cell cycle, as well as the aging and apoptosis of livingorganisms are mostly a result of selective expression of specific geneswithin cells. This is true for cellular mechanisms associated withdiseases. Particularly, pathological phenomena, such as oncogenesis, areinduced by gene mutations that in the end lead to changes in geneexpression.

ONC201 (7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido [3,4-e]pyrimidin-5(1H)-one) is the founding member of aclass of anti-cancer compounds called imipridones that is in Phase IIclinical trials in multiple advanced cancers. Since the discovery ofONC201 as a p53-independent inducer of TRAIL gene transcription,preclinical studies have determined that ONC201 has anti-proliferativeand pro-apoptotic effects against a broad range of tumor cells but notnormal cells. The mechanism of action of ONC201 involves engagement ofPERK-independent activation of the integrated stress response, leadingto tumor upregulation of DR5 and dual Akt/ERK inactivation, andconsequent Foxo3a activation leading to upregulation of the death ligandTRAIL. ONC201 is orally active with infrequent dosing in animal models,causes sustained pharmacodynamic effects, and is not genotoxic. Thefirst-in-human ONC201 clinical trial in advanced aggressive refractorysolid tumors confirmed that it is well-tolerated. In summary, theimipridone family that comprises ONC201 and its chemical analogsrepresent a new class of therapeutic agents.

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided herein are compounds of formula (10):

wherein R₁ and R₂ are independently selected from H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl,aralkoxy, aralkylthio, and acyl radicals. In one embodiment, when R₁ isCH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂is CH₂-(2-CH₃-Ph) (i.e., ONC201). In one embodiment, R₁ is CH₂Ph and R₂is CH₂-(2,4-di F-Ph) (i.e., ONC206). In one embodiment, R₁ is CH₂Ph andR₂ is CH₂-(4-CF₃-Ph) (i.e., ONC212). In one embodiment, R₁ is CH₂Ph andR₂ is CH₂-(3,4-di F-Ph) (i.e., ONC213). In one embodiment, R₁ is CH₂(3,4-di-Cl-Ph and R₂ is CH₂-(4-CF₃-Ph) (i.e., ONC234). In oneembodiment, R₁ is CH₂-3-thienyl and R₂ is CH₂-(4-CF₃-Ph) (i.e., ONC236).

In another aspect, provided herein are methods of treating or preventinga disease, disorder, or condition in a subject in need thereof,comprising: administering to the subject in need of such treatment apharmaceutical composition comprising a therapeutically effective amounta compound of formula (10) or an analog thereof, or a pharmaceuticallyacceptable salt thereof. In one embodiment, the compound is selectedfrom the group consisting of ONC201, ONC206, ONC212, ONC213, ONC234 andONC236. In one embodiment, the subject has, or is at risk of having,cancer. In one embodiment, the cancer is selected from a central nervoussystem tumor, a brain tumor, a peripheral nervous system tumor, apheochromocytoma, a paraganglioma, a neuroendocrine tumor, ewingssarcoma, a pancreatic cancer, a prostate cancer, an endometrial cancer,a hematological malignancy, a bone cancer, and a lymphatic system tumor.In one embodiment, the cancer is selected from meningioma, ependymoma,glioma, neuroblastoma, or diffuse intrinsic pontine glioma. In oneembodiment, the cancer is selected from an acute leukemia selected froman acute lymphocyte leukemia, acute myeloid leukemia, myelodysplasticsyndrome, or myeloproliferative disease. In one embodiment, In oneembodiment, the cancer has a histone H3 mutation (e.g., the mutationH3.3 K27M) or an epigenetically silenced unmethylatedO(6)-methylguanine-DNA methyltransferase (MGMT) gene. In one embodiment,the subject has, or is at risk of having, a psychiatric disorder. In oneembodiment, the psychiatric disorder is selected from a psychosis,schizophrenia, bipolar disorder, or major depressive disorder. In oneembodiment, the subject has, or is at risk of having, an infection. Inone embodiment, the infection is a bacterial infection. In oneembodiment, the infection is a gram-negative bacterial infection. In oneembodiment, the infection is a gram-positive bacterial infection. In oneembodiment, the bacterial infection is an infection of a bacteriaselected from the group consisting of Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,Pseudomonas aeruginosa, and Enterobacter species. In one embodiment, thebacterial infection is a Staphylococcus infection. In one embodiment,the Staphylococcus infection is an S. aureus infection (e.g., amethicillin-resistant S. aureus (MRSA) infection).

In another aspect, provided herein are methods of treating or preventinga disease, disorder, or condition in a subject in need of selectivemodulation of the activity of a G protein-coupled receptor (GPCR) or ofa G protein-coupled receptor (GPCR) signaling pathway. Modulationincludes, but is not limited to, agonism, partial agonism, inverseagonism, partial antagonism, antagonism, bivalent modulation, or bitopicmodulation. In one embodiment, the methods comprise administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof. Inone embodiment, the subject has, or is at risk of having, cancer. In oneembodiment, the subject has, or is at risk of having, a psychiatricdisorder. In one embodiment, the psychiatric disorder is psychosis. Inone embodiment, the psychiatric disorder is schizophrenia. In oneembodiment, the subject has, or is at risk of having, an infection. Inone embodiment, the infection is a bacterial infection. In oneembodiment, the infection is a gram-negative bacterial infection. In oneembodiment, the infection is a gram-positive bacterial infection. In oneembodiment, the bacterial infection is an infection of a bacteriaselected from Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, orEnterobacter species. In one embodiment, the bacterial infection is aStaphylococcus infection. In one embodiment, the Staphylococcusinfection is an S. aureus infection (e.g., a methicillin-resistant S.aureus (MRSA) infection). In one embodiment, the treatment regimencomprises administering an effective amount of a therapeutic, such ascompound of formula (10), a pharmaceutically acceptable salt thereof, oran analog thereof. In one embodiment, the GPCR is a Class A GPCR. In oneembodiment, the GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3,LGR4, PTGIR, CXCR7 or a combination thereof. In one embodiment, the GPCRis GPR132 (also called G2A). In one embodiment, the GPCR is GPR91. Inone embodiment, the GPCR is MTNR1A. In one embodiment, the GPCR isCXCR7.

In another aspect, provided herein are methods of treating or preventinga disease, disorder, or condition in a subject in need of selectivemodulation of the activity of a dopamine receptor or of a member of adopamine receptor signaling pathway. In one embodiment, the methodscomprise administering to the subject in need of such treatment apharmaceutical composition comprising a therapeutically effective amounta compound of formula (10) or an analog thereof, or a pharmaceuticallyacceptable salt thereof. In one embodiment, the subject has, or is atrisk of having, cancer. In one embodiment, the subject has, or is atrisk of having, a psychiatric disorder. In one embodiment, thepsychiatric disorder is psychosis. In one embodiment, the psychiatricdisorder is schizophrenia. In one embodiment, the subject has, or is atrisk of having, an infection. In one embodiment, the infection is abacterial infection. In one embodiment, the infection is a gram-negativebacterial infection. In one embodiment, the infection is a gram-positivebacterial infection. In one embodiment, the bacterial infection is aninfection of a bacteria selected from the group consisting ofEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspecies. In one embodiment, the bacterial infection is a Staphylococcusinfection. In one embodiment, the Staphylococcus infection is an S.aureus infection (e.g., a methicillin-resistant S. aureus (MRSA)infection). In one embodiment, the treatment regimen comprisesadministering an effective amount of a therapeutic, such as compound offormula (10), a pharmaceutically acceptable salt thereof, or an analogthereof. In one embodiment, the dopamine receptor is from the D2-likefamily of dopamine receptors.

In another aspect, provided herein are methods of treating or preventingliver fibrosis or of regenerating liver tissue, comprising:administering to the subject in need of such treatment a pharmaceuticalcomposition comprising a therapeutically effective amount a compound offormula (10) or a compound of formula (100) (e.g., TIC-10), or an analogthereof, or a pharmaceutically acceptable salt thereof. In oneembodiment, the compound is a CXCR7 agonist.

In another aspect, provided herein are methods of stimulating the immunesystem (e.g., activating NK cells) in a subject in need thereof,comprising: administering to the subject a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof. Inone embodiment, the compound is a GPR91 agonist. In one embodiment, thecompound is ONC213. In one embodiment, the subject has cancer and themethod is a method of cancer immunotherapy. In one embodiment, thesubject has a viral infection (e.g., HIV). In one embodiment, thesubject has systemic lupus erythematosus. In one embodiment, the methodfurther comprises administering a vaccine (e.g., a cancer vaccine) tothe subject, and the compound is administered as an adjuvant.

In another aspect, provided herein are methods of identifying whether asubject having a condition is likely to be responsive to a treatmentregimen described herein. In one embodiment, the methods comprise (i)obtaining a biological sample from the subject; (ii) measuringexpression levels of at least one dopamine receptor or G protein-coupledreceptor (GPCR) in the sample; (iii) comparing the levels measured inthe sample to those for a pre-determined standard; and (iv) determiningwhether the subject is likely to be responsive to the treatment regimen,based on the levels measured in the sample to those for thepre-determined standard. In one embodiment, the subject has, or is atrisk of having, cancer. In one embodiment, the subject has, or is atrisk of having, a psychiatric disorder. In one embodiment, the subjecthas, or is at risk of having, an infection. In one embodiment, thetreatment regimen further comprises administering an effective amount ofa therapeutic, such as compound of formula (10), a pharmaceuticallyacceptable salt thereof, or an analog thereof. In one embodiment, thedopamine receptor is from the D2-like family of dopamine receptors. Inone embodiment, the GPCR is a Class A GPCR. In one embodiment, the GPCRis GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7 or acombination thereof. In one embodiment, the GPCR is GPR132, GPR91,MTNR1A, CXCR7 or a combination thereof. In one embodiment, the GPCR isGPR132.

In another aspect, provided herein are methods of assessing theeffectiveness of a treatment regimen described herein, monitoring, orproviding a prognosis for a subject with a condition. In one embodiment,the methods comprises (i) obtaining a biological sample from thesubject; (ii) measuring expression levels of at least one dopaminereceptor or G protein-coupled receptor (GPCR) in the sample; (iii)comparing the levels measured in the sample to those for apre-determined standard; and (iv) determining a prognosis or determiningwhether the subject is responsive to the treatment regimen, based on thelevels measured in the sample to those for the pre-determined standard.In one embodiment, the methods comprises (i) obtaining a biologicalsample from the subject; (ii) measuring gene copy number or mutations inat least one dopamine receptor in the sample; (iii) comparing the copynumber measured or mutations found in the sample to those for apre-determined standard; and (iv) determining whether the subject isresponsive to the treatment regimen, based on the copy number measuredor mutations found in the sample to those for the pre-determinedstandard. In one embodiment, the subject has, or is at risk of having,cancer. In one embodiment, the subject has, or is at risk of having, apsychiatric disorder. In one embodiment, the subject has, or is at riskof having, an infection. In one embodiment, the treatment regimencomprises administering an effective amount of a therapeutic, such ascompound of formula (10), a pharmaceutically acceptable salt thereof, oran analog thereof. In one embodiment, the dopamine receptor is selectedfrom DRD2, DRD2S, DRD2L, and DRD3. In one embodiment, the dopaminereceptor is from the D2-like family of dopamine receptors In oneembodiment, the GPCR is a Class A GPCR. In one embodiment, the GPCR isGPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7 or acombination thereof.

In another aspect, provided herein are methods for screening a potentialtherapeutic for a condition. In one embodiment, the method comprises (i)contacting at least one G protein-coupled receptor (GPCR) with a testmolecule suspected of being a therapeutic for a condition; (ii)measuring the binding affinity, interaction or GPCR signalling of thetest compound to the GPCR; and (iii) comparing the binding affinity,interaction or signalling of the test molecule to a pre-determinedthreshold. In one embodiment, GPCR modulation or GPCR signalingmodulation by the test molecule comparable to or greater than thethreshold is indicative of a therapeutic for the condition. In oneembodiment, the condition is cancer. In one embodiment, thepre-determined threshold is the GPCR modulation or GPCR signalingmodulation of a therapeutic, such as a compound of formula (10) or apharmaceutically acceptable salt thereof, or an analog thereof. In oneembodiment, the GPCR is a Class A GPCR. In one embodiment, the GPCR isGPR132. In one embodiment, the GPCR is GPR132, GPR91, MTNR1A, GPR162,GPR137, BAI3, LGR4, PTGIR, CXCR7 or a combination thereof. In oneembodiment, the GPCR is GPR132. In one embodiment, the GPCR is GPR91. Inone embodiment, the GPCR is MTNR1A. In one embodiment, the GPCR isCXCR7.

In another aspect, provided herein are methods for screening a potentialtherapeutic for a condition. In one embodiment, the method comprises (i)contacting at least one dopamine receptor with a test molecule suspectedof being a therapeutic for a condition; (ii) measuring the bindingaffinity, interaction or signalling of the test molecule to the at leastone dopamine receptor; and (iii) comparing the binding affinity orinteraction of the test molecule to a pre-determined threshold. In oneembodiment, modulation of the dopamine receptor by the test moleculecomparable to or greater than the threshold is indicative of atherapeutic for the condition. In one embodiment, the condition iscancer. In one embodiment, the dopamine receptor is a member of theD2-like family of dopamine receptors. In one embodiment, thepre-determined threshold is the modulation of the dopamine receptor ordopamine receptor signalling by a therapeutic, such as a compound offormula (10) or a pharmaceutically acceptable salt thereof, or an analogthereof.

In another aspect, provided herein are methods for screening a potentialtherapeutic for a condition. In one embodiment, using a processor, themethod comprises (i) using a computational docking method to modelbinding or interaction, if any, of one or more 3-dimensional structures(conformations) of a test molecule suspected of being a therapeutic forthe condition to a 3-dimensional structure or model of at least onedopamine receptor; (ii) using the computational method to estimate thebinding affinity or interaction of the test molecule structure to thestructure or model of the at least one dopamine receptor; and (iii)using the computational method to compare the binding affinity orinteraction of the test molecule to a pre-determined threshold, whereinmodulation of the dopamine receptor by the test molecule comparable toor greater than the threshold is indicative of a therapeutic for thecondition. In one embodiment, the condition is cancer. In oneembodiment, the dopamine receptor is a member of the D2-like family ofdopamine receptors.

In another aspect, provided herein are methods of treating a subjecthaving a condition. In one embodiment, the method comprisesadministering an effective amount of a therapeutic agent that targets atleast one dopamine receptor or G protein-coupled receptor (GPCR). In oneembodiment, the therapeutic agent is a neutralizing agent. In oneembodiment, the therapeutic agent is an antagonist of the receptor. Inone embodiment, the therapeutic agent is an agonist of the receptor. Inone embodiment, the therapeutic agent is a competitive inhibitor of thereceptor with respect to dopamine. In one embodiment, the therapeuticagent is a non-competitive inhibitor of the receptor with respect todopamine. In one embodiment, the therapeutic agent is selective for theD2-like family of dopamine receptors with respect to the D1-like familyof dopamine receptors. In one embodiment, the subject has, or is at riskof having, cancer. In one embodiment, the subject has, or is at risk ofhaving, a psychiatric disorder. In one embodiment, the subject has, oris at risk of having, an infection. In one embodiment, the dopaminereceptor is a member of the D2-like family of dopamine receptors. In oneembodiment, the GPCR is a Class A GPCR. In one embodiment, the GPCR isGPR132. In one embodiment, the GPCR is GPR91. In one embodiment, theGPCR is MTNR1A. In one embodiment, the GPCR is CXCR7. In one embodiment,the GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR,CXCR7 or a combination thereof. In one embodiment, the therapeutic agentis a monoclonal antibody (e.g., a chimerized or humanized monoclonalantibody), polyclonal antibody (e.g., a chimerized or humanizedpolyclonal antibody), or a bispecific antibody. In one embodiment, thetherapeutic agent is a drug or active agent, such as an anti-canceragent, conjugated to an antibody. In one embodiment, the therapeuticagent is a radioactively-conjugated antibody or a smallmolecule-conjugated antibody. In one embodiment, the therapeutic agentis a vector that expresses a recombinant antibody to the dopaminereceptor or GPCR. In one embodiment, the therapeutic agent is a fusionprotein or a peptide that targets the dopamine receptor or GPCR. In oneembodiment, the therapeutic agent is an siRNA, shRNA, or an antisenseoligonucleotide that targets the dopamine receptor or GPCR. In oneembodiment, the dopamine receptor or GPCR is targeted by CRISPRinterference.

In another aspect, provided herein are methods of treating and assessingthe efficacy of a treatment in a subject having a condition. In oneembodiment, the method comprises (i) treating the subject according to atreatment method described herein (ii) assessing as described herein thetreatment's efficacy. In one embodiment, the subject has, or is at riskof having, cancer. In one embodiment, the treatment regimen comprisesadministering an effective amount of a therapeutic, such as a compoundof formula (10) or a pharmaceutically acceptable salt thereof or ananalog thereof. In one embodiment, the dosage of a therapeuticadministered, the frequency of administration of the compound (e.g., acompound of formula (10)), or both, is selected or adjusted based on thelevels of gene expression or gene copy number measured or mutationsfound.

BRIEF DESCRIPTION OF THE DRAWINGS

The above summary, as well as the following detailed description ofembodiments of the invention, will be better understood when read inconjunction with the appended drawings. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities shown. In the drawings:

FIG. 1. Antagonism of dopamine receptors (DRD1, DRD2S, DRD2L, DRD3,DRD4, and DRD5) by ONC201.

FIG. 2 illustrates soluble prolactin detected by ELISA in the peripheralblood of advanced solid tumor patients at baseline and following asingle ONC201 dose (PO 125-625 mg). Sampling time points post-treatmentinclude 6 hours, 1, 2, 7, and 21 days post-treatment.

FIG. 3. Tumor type sensitivity of the Genomic of Drug Sensitivity inCancer program (GDSC) cell line collection. The average sensitivity wasdetermined by average estimated IC₅₀ values from cell viability assaysconducted at 72 hours post-treatment. Numbers above the bar indicatesindicate the number of cell lines per tumor type.

FIG. 4. ONC201 is a selective DRD2 antagonist. (A) Agonism of orphan orknown GPCRs or antagonism of known GPCRs using an arrestin recruitmentreporter assay (10 μM ONC201). (B) Antagonism of ligand-stimulateddopamine receptors by ONC201 using an arrestin recruitment reporterassay. Schild analysis of DRD2L antagonism by ONC201 using (C) arrestinrecruitment or (D) cAMP modulation reporters.

FIG. 5. ONC201 antagonism of DRD2 is highly specific among GPCRs andother cancer drug targets. (A) Antagonism of GPCRs using an arrestinrecruitment reporter assay (10 μM ONC201). Competition ofONC201-mediated antagonism of DRD2L by dopamine in (B) arrestinrecruitment or (C) cAMP modulation reporters. (D) Antagonism or agonismof nuclear hormone receptors by ONC201 (2 or 20 μM) with a nucleartranslocation reporter assay. (E) In vitro inhibition of kinaseenzymatic activity by ONC201 (1 μM). (F) DRD2L antagonistic activity ofONC201 or an ONC201 linear isomer with no biological activity using anarrestin recruitment reporter assay.

FIG. 6. GBM cell lines with higher DRD2 expression are more responsiveto ONC201. (A) Inhibition of NCI60 GBM cell lines as a function ofONC201 concentration. (B) Log ONC201 GI₅₀ (M) vs DRD2 expression foreach GBM cell line. R²=0.8707.

FIG. 7. ONC201 exhibits superior selectivity among GPCRs for DRD2compared to other DRD2 antagonists, such as risperidone.

FIG. 8. ONC201 has a higher selectivity for tumor cells than theantipsychotic DRD2 antagonist, thioridazine.

FIG. 9. Optimization of ONC201 inhibition of DRD2 calcium flux. HEK-293Tcells were transfected with expression constructs for wild-type DRD2 (A)or a control GPCR (B). DRD2-specific calcium flux inhibition wasinvestigated at 0.1 and 1 nM dopamine, for ONC201 concentrations between100 pM and 100 μM. 100 μM ONC201 completely inhibited DRD2dopamine-induced calcium flux but had no effect on the control GPCR.

FIG. 10. Comparison of DRD2 inhibitors. DRD2-specific calcium fluxinhibition was investigated at 1 nM dopamine, using inhibitors spiperone(squares), domperidone (triangles), and ONC201 (circles) at a range ofconcentrations. Data for individual assays was normalized using theno-inhibitor value (shown as 10⁻¹¹ M) as 100% activity.

FIG. 11. Identification of DRD2 residues critical for dopamine-inducedcalcium flux. (A) Dopamine-induced calcium flux was assayed as before at1 nM dopamine, across the entire DRD2 alanine-scan library. The datarepresent the average of three experiments. Mutant clones wereconsidered to be deficient for calcium flux if they demonstrated fluxvalues less than 2 standard deviations below the average calcium fluxvalue (AV−2SD) for the entire library. (B) The locations of the 28mutated residues identified are indicated (green spheres) on the DRD3crystal structure (PDB id 3PBL; Chien, E. Y. et al. (2010) Science330:1091-5). The D2R/D3R antagonist eticlopride is shown in cyan.

FIG. 12. Identification of DRD2 residues critical for ONC201 inhibitionof dopamine-induced calcium flux. (A) Dopamine-induced calcium flux wasassayed as before at 1 nM dopamine but in the presence of 100 μM ONC201,across the entire DRD2 alanine-scan library. The data represent theaverage of three experiments normalized to the value for flux value withwild-type DRD2 (% WT). Mutant clones were considered to be critical forONC201 inhibition if they demonstrated flux values greater than 2standard deviations above the average calcium flux value (AV+2SD) forthe entire library. (B) The locations of the 8 mutated residuesidentified are indicated (red spheres) on the DRD3 crystal structure.

FIG. 13. A reference compound, (+) Butaclamol, and a test compound,ONC201 dihydrochloride, successfully competed for [³H]Methylspiperone,with IC₅₀ values of 2.5 nM and 21 μM, respectively.

FIG. 14. Association kinetic curves for ONC201 dihydrochloride to DRD2Sreceptor to determine K_(on) and K_(off).

FIG. 15. Compound activity with the selected GPCR and Orphan GPCRBiosensor Assays. Compound was tested in antagonist and agonist modewith the desired GPCR and Orphan GPCR Biosensor Assays. For agonistassays, data was normalized to the maximal and minimal response observedin the presence of control ligand and vehicle. For antagonist assays,data was normalized to the maximal and minimal response observed in thepresence of EC₈₀ ligand and vehicle. The following EC₈₀ concentrationswere used: CCR4 Arrestin: 0.0078 μM CCL22; CHRM2 Arrestin: 26 μMAcetylcholine; and MC4R Arrestin: 0.0026 μM Melanotan II.

FIG. 16. ONC206 and ONC212 demonstrated anti-cancer efficacy acrossvarious tumor types in the NCI60 cancer cell line panel. ONC203 is aninactive negative control

FIG. 17. ONC206 is an imipridone with improved DRD2 antagonism. ONC206,an analog of ONC201, exhibits superior antagonism of D2-like dopaminereceptor family, and retains highly selective antagonism of D2-likedopamine receptors compared to other antipsychotics, such as ahaloperidol.

FIG. 18. Bone cancer is more responsive to ONC206 than ONC201.

FIG. 19. Ewing's sarcoma is the most ONC206 responsive bone cancersubtype.

FIG. 20. ONC206 anti-cancer efficacy is in the nanomolar range in 14 outof 16 Ewing's sarcoma cell lines. ONC206 demonstrated superior efficacycompared to ONC201 in all cell lines

FIG. 21. The imipridone ONC212 targets an orphan GPCR It is a highlyselective agonist of the orphan GPCR tumor suppressor GPR132, and itdoes not engage DRD2.

FIG. 22. ONC212 induced cell death in cancer cells (HCT116) but notnormal cells (MRC5) at nanomolar concentrations.

FIG. 23. ONC212 induces the integrated stress response and inhibitsAkt/ERK phosphorylation at nanomolar concentrations and at earlier timepoints compared to ONC201.

FIG. 24. ONC212 demonstrates oral and IP anti-cancer efficacy inxenograft mouse models of colorectal and breast cancer.

FIG. 25. Leukemia is more responsive to ONC212 than ONC201.

FIG. 26. ONC212 demonstrates anti-cancer efficacy (and superior efficacycompared to ONC201) in the nanomolar range in 55 leukemia cell linesregardless of subtype.

FIG. 27. GPCRs agonized or antagonized (>50%) by 9 imipridones tested.Imipridones selectively target rhodopsin-like Class A GPCRs.

FIG. 28. Case study of a subject with recurrent glioblastoma (Example16). (A) Tumor size relative to baseline (%) of total tumor burden inthe subject. One cycle is 3 weeks. (B) Contrast MRI scans at baseline,21, 27 and 36 weeks post-ONC201 initiation of one of 2 malignantlesions.

FIG. 29. ONC212 demonstrates anti-cancer effects in acute myeloidleukemia (AML) cell lines. (A) Comparison of cell viability of MV411 AMLcells treated with ONC212 or cytarabine. (B) Comparison of cellviability of MOLM14, MV411 AML cells, MRC5 lung fibroblasts and Hs27abone marrow cells treated with ONC212. (C) Cell viability of MOLM14 andMV411 AML cells treated with ONC212 (250 nM) for 4, 8, 24, 48, 72 and 96h.

FIG. 30. ONC212 efficacy in ONC201-resistant AML xenograft model (MV411AML cells (5×10⁶) subcutaneously implanted in the flanks of athymic nudemice). ONC212 and ONC201 were administered orally (PO) as indicated.Tumor volume (A and B) and body weight (C) (n=10) was measured onindicated days. * represents p<0.05 relative to vehicle.

FIG. 31. ONC206 efficacy in Ewing's sarcoma xenograft model (MHH-ES-1Ewing's sarcoma cells (5×10⁶) subcutaneously implanted in the flanks ofathymic nude mice). ONC206 (PO) and methotrexate (IV) were administeredon day 1 and day 13 as indicated. Tumor volume (A) and body weight (B)(n=4) was measured on indicated days.

FIG. 32. ONC213 (10 μM) GPCR profile using a β-arrestin recruitmentreporter assay.

FIG. 33. ONC213 demonstrated in vitro anti-cancer potency inHCT116/RPMI8226 cancer cells similar to ONC212, but in vitro toxicity tonormal cells was reduced compared to ONC212.

FIG. 34. ONC237 (10 μM) GPCR profile using a β-arrestin recruitmentreporter assay.

FIG. 35. ONC236 (10 μM) GPCR profile using a β-arrestin recruitmentreporter assay.

FIG. 36. ONC234 (10 μM) GPCR profile using a β-arrestin recruitmentreporter assay.

FIG. 37. ONC201 linear isomer (TIC-10) (10 μM) GPCR profile using aβ-arrestin recruitment reporter assay.

FIG. 38. Number of GPCRs hit for several imipridones.

DETAILED DESCRIPTION OF THE INVENTION

Scientific and technical terms used here are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found and used in context in various standard referencesillustratively including J. Sambrook and D. W. Russell, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press;3^(rd) Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts of RNAiTechnology, DNA Press LLC, Eagleville, P A, 2003; Herdewijn, P. (Ed.),Oligonucleotide Synthesis: Methods and Applications, Methods inMolecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K.Vintersten, R. Behringer, Manipulating the Mouse Embryo: A LaboratoryManual, 3^(rd) edition, Cold Spring Harbor Laboratory Press; Dec. 15,2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells:methods and protocols in Methods Mol Biol. 2002; 185, Humana Press;Current Protocols in Stem Cell Biology, ISBN: 9780470151808, as well asU.S. Pat. No. 8,673,923. The content of each of the references above ishereby incorporated by reference in its entirety.

The term “substituted” means that any one or more hydrogens on thedesignated atom is replaced with a selection from the indicated group,provided that the designated atom's normal valency is not exceeded, andthat the substitution results in a stable compound. When a substituentis keto (i.e., ═O), then 2 hydrogens on the atom are replaced. Ketosubstituents are not present on aromatic moieties. Ring double bonds aredouble bonds that are formed between two adjacent ring atoms (e.g., C═C,C═N, or N═N).

When a variable (e.g., R⁴) occurs more than one time in a constituent orformula for a compound, its definition at each occurrence is independentof its definition at every other occurrence. Thus, for example, if agroup is shown to be substituted with 0-3 R⁴ moieties, then the groupmay optionally be substituted with up to three R⁴ moieties and R⁴ ateach occurrence is selected independently from the definition of R⁴.Also, combinations of substituents and/or variables are permissible, butonly if such combinations result in stable compounds.

When an atom or chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it will be appreciated that this is meant toencompass each number within the range, as well as all intermediateranges. For example, “C₁₋₆ alkyl” is meant to include alkyl groups with1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5,3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” includes both branched and straight-chain saturatedaliphatic hydrocarbon groups having the specified number of carbonatoms. For example, C₁₋₆ alkyl is intended to include C₁, C₂, C₃, C₄,C₅, and C₆ alkyl groups. Examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl s-butyl,t-butyl, n-pentyl, s-pentyl, neopentyl and n-hexyl. In certain cases, astraight chain or branched chain alkyl has six or fewer carbon atoms inits backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain),and in other cases, a straight chain or branched chain alkyl has four orfewer carbon atoms. Likewise, cycloalkyls have from three to eightcarbon atoms in their ring structure, and in other cases, cycloalkylshave five or six carbons in the ring structure. Most preferred is C₁₋₆alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl,n-hexyl and cyclopropylmethyl.

the term “substituted alkyl” means alkyl as defined above, substitutedby one, two or three substituents selected from halogen, —OH, alkoxy,—NH₂, —N(CH₃)₂, —C(═O)OH, trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl,—C(═O)NH₂, —SO₂NH₂, —C(═NH)NH₂, and —NO₂, preferably containing one ortwo substituents selected from halogen, —OH, alkoxy, —NH₂,trifluoromethyl, —N(CH₃)₂, and —C(═O)OH, more preferably selected fromhalogen, alkoxy and —OH. Examples of substituted alkyls include, but arenot limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and3-chloropropyl.

Unless the number of carbons is otherwise specified, “lower alkyl” is analkyl group, as defined above, but having from one to six carbon atoms,preferably one to four, in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of 2-6 carbon atoms and preferably2-4 carbon atoms.

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includesstraight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched-chainalkenyl groups, cycloalkenyl (e.g., alicyclic) groups (e.g.,cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certain cases,a straight chain or branched chain alkenyl group has six or fewer carbonatoms in its backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ forbranched chain). Likewise, cycloalkenyl groups may have from three toeight carbon atoms in their ring structure, and in some embodiments,cycloalkenyl groups have five or six carbons in the ring structure. Theterm “C₂-C₆” includes alkenyl groups containing two to six carbon atoms.The term “C₃-C₆” includes alkenyl groups containing three to six carbonatoms.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight-chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), branched-chain alkynyl groups, andcycloalkyl or cycloalkenyl substituted alkynyl groups. In certainembodiments, a straight chain or branched chain alkynyl group has six orfewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “cycloalkyl” refers to a mono cyclic or polycyclic non-aromaticradical, where each of the atoms forming the ring (i.e. skeletal atoms)is a carbon atom. In some cases, the cycloalkyl group is saturated orpartially unsaturated. In other cases, the cycloalkyl group is fusedwith an aromatic ring. Cycloalkyl groups include groups with from 3 to10 ring atoms. Examples of cycloalkyl groups include, but are notlimited to, the following moieties:

Monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include,but are not limited to, tetrahydronaphthyl, indanyl, andtetrahydropentalene. Polycyclic cycloalkyls include adamantine andnorbornane. The term cycloalkyl includes “unsaturated nonaromaticcarbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both ofwhich refer to a nonaromatic carbocycle as defined herein, whichcontains at least one carbon carbon double bond or one carbon carbontriple bond.

The term “cycloalkylalkyl” refers to an alkyl group substituted by acycloalkyl group. Example cycloalkylalkyl groups includecyclopropylalkyl, cyclohexylalkyl.

The term “heterocycloalkyl” refers to a non-aromatic heterocycle whereone or more of the ring-forming atoms is a heteroatom such as an O, N,or S atom. Heterocycloalkyl groups include mono- or polycyclic (e.g.,having 2, 3 or 4 fused rings) ring systems, as well as spirocycles.Example heterocycloalkyl groups include morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, and imidazolidinyl. Also included in thedefinition of heterocycloalkyl can be moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thenonaromatic heterocyclic ring, for example quinolyl, isoquinolyl, andbenzo derivatives of heterocycles. A heterocycloalkyl group having oneor more fused aromatic rings are attached though either the aromatic ornon-aromatic portion. Also included in the definition ofheterocycloalkyl are moieties where one or more ring-forming atoms canbe substituted by 1 or 2 oxo or sulfido groups. In some cases, theheterocycloalkyl group has from 1 to about 20 carbon atoms, and infurther case from about 3 to about 20 carbon atoms. In some cases, aheterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about7, or 5 to 6 ring-forming atoms. In some cases, a heterocycloalkyl grouphas 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some cases, aheterocycloalkyl group contains 0 to 3 double bonds. In some cases, aheterocycloalkyl group contains 0 to 2 triple bonds.

The term “heterocycloalkylalkyl” refers to an alkyl group substituted bya heterocycloalkyl. Example heterocycloalkylalkyls includemorpholinoalkyl and piperazinylalkyl.

The term “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or4 fused rings) aromatic hydrocarbons such as, phenyl, naphthyl,anthracenyl, phenanthrenyl. In some cases, an aryl group has from 6 toabout 20 carbon atoms.

The term “arylalkyl” refers to an alkyl group substituted by an arylgroup. Example arylalkyl groups include benzyl and phenylethyl.

The term “heteroaryl” refers to an aromatic heterocycle having at leastone heteroatom ring member such as an O, S, or N atom. Heteroaryl groupsinclude monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings)systems. A ring-forming N atom in a heteroaryl group can also beoxidized to form an N-oxo moiety. Examples of heteroaryl groups includepyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl,pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl,isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl,indolinyl. In some cases, a heteroaryl group has from 1 to about 20carbon atoms, and in further cases from about 3 to 20 carbon atoms. Insome cases, a heteroaryl group contains 3 to about 14, 3 to about 7, or5-6 ring-forming atoms. In some cases, a heteroaryl group has 1 to about4, 1 to about 3, or 1-2 heteroatoms.

a “heteroarylalkyl” group refers to an alkyl group substituted by aheteroaryl group. An example of a heteroarylalkyl group ispyridylmethyl.

The terms “halo” or “halogen” refer to a fluorine (F), chlorine (Cl),bromine (Br), or iodine (I) atom; preferably, F, Cl, or Br; morepreferably, F or Cl. The term “perhalogenated” refers to a moiety whereall hydrogens are replaced by halogens. The term “haloalkyl” refers toalkyl moieties with a halogen replacing a hydrogen on one or morecarbons of the hydrocarbon backbone. C₁-C₆ haloalkyl includes a straightchain or branched alkyl with six or fewer backbone carbon atoms and ahalogen replacing a hydrogen on one or more backbone carbons.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.C₁-C₆ alkoxy refers to moieties having six or fewer carbon atoms in thehydrocarbon backbone. Examples of alkoxy groups (or alkoxyl radicals)include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxygroups. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.Examples of substituted alkoxy groups include halogenated alkoxy groups.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “pharmaceutically acceptable salts” refers to derivatives ofcompounds where the parent compound is modified by converting anexisting acid or base moiety to its salt form. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids.Pharmaceutically acceptable salts include conventional non-toxic saltsof a parent compound formed, for example, from non-toxic inorganic ororganic acids. Pharmaceutically acceptable salts may be synthesized froma parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting a free acid or base form of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts can be found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Journal of Pharmaceutical Science, 66, 2 (1977), andP. H. Stahl and C. G. Wermuth, editors, Handbook of PharmaceuticalSalts: Properties, Selection and Use, 2^(nd) Revised edition,Weinheim/Zürich: Wiley-VCH/VHCA (2011), each of which is incorporatedherein by reference in its entirety.

Examples of suitable inorganic acids include hydrochloric acid,sulphuric acid, phosphoric acid, or hydrobromic acid, while examples ofsuitable organic acids include carboxylic acid, sulpho acid, orsulphonic acid, such as acetic acid, tartaric acid, lactic acid,propionic acid, glycolic acid, malonic acid, maleic acid, fumaric acid,tannic acid, succinic acid, alginic acid, benzoic acid, 2-phenoxybenzoicacid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid,maleic acid, salicylic acid, trifluoroacetic acid, 3-aminosalicylicacid, ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid,oxalic acid, gluconic acid, amino acids, methanesulphonic acid,ethanesulphonic acid, 2-hydroxyethanesulphonic acid,ethane-1,2-disulphonic acid, benzenesulphonic acid,4-methylbenzenesulphonic acid or naphthalene-2-sulphonic acid. Examplesof suitable inorganic bases include sodium hydroxide, potassiumhydroxide and ammonia, while examples of suitable organic bases includeamines, e.g., tertiary amines, such as trimethylamine, triethylamine,pyridine, N,N-dimethylaniline, quinoline, isoquinoline, α-picoline,γ-picoline, quinaldine, or pyrimidine.

the term “antibody” encompasses the structure that constitutes thenatural biological form of an antibody. In most mammals, includinghumans, and mice, this form is a tetramer and consists of two identicalpairs of two immunoglobulin chains, each pair having one light and oneheavy chain, each light chain comprising immunoglobulin domains V_(L)and C_(L), and each heavy chain comprising immunoglobulin domains V_(H),Cγ1, Cγ2, and Cγ3. In each pair, the light and heavy chain variableregions (V_(L) and V_(H)) are together responsible for binding to anantigen, and the constant regions (C_(L), Cγ1, Cγ2, and Cγ3,particularly Cγ2, and Cγ3) are responsible for antibody effectorfunctions. In some mammals, for example in camels and llamas,full-length antibodies may consist of only two heavy chains, each heavychain comprising immunoglobulin domains V_(H), Cγ2, and Cγ3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genesImmunoglobulins include but are not limited to antibodiesImmunoglobulins may have a number of structural forms, includingfull-length antibodies, antibody fragments, and individualimmunoglobulin domains including V_(H), Cγ1, Cγ2, Cγ3, V_(L), and C_(L).

Based on the heavy-chain constant domain amino acid sequence, intactantibodies can be assigned to different “classes.” There are five-majorclasses (isotypes) of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into “subclasses,” e.g.,IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domainsthat correspond to the different antibody classes are called alpha,delta, epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known to one skilled in the art.

The terms “antibody” or “antigen-binding fragment,” respectively, referto intact molecules as well as functional fragments thereof, such asFab, a scFv-Fc bivalent molecule, F(ab′)₂, and Fv that are capable ofspecifically interacting with a desired target. In some cases, theantigen-binding fragments comprise:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule, which can be        produced by digestion of whole antibody with the enzyme papain        to yield an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule that can be        obtained by treating whole antibody with pepsin, followed by        reduction, to yield an intact light chain and a portion of the        heavy chain; two Fab′ fragments are obtained per antibody        molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, a genetically engineered fragment containing the        variable region of the light chain and the variable region of        the heavy chain expressed as two chains;    -   (5) Single chain antibody (“SCA”), a genetically engineered        molecule containing the variable region of the light chain and        the variable region of the heavy chain, linked by a suitable        polypeptide linker as a genetically fused single chain molecule;        and    -   (6) scFv-Fc, is produced by fusing single-chain Fv (scFv) with a        hinge region from an immunoglobulin (Ig) such as an IgG, and Fc        regions.

In one embodiment, an antibody provided herein is a monoclonal antibody.In one embodiment, the antigen-binding fragment provided herein is asingle chain Fv (scFv), a diabody, a tandem scFv, a scFv-Fc bivalentmolecule, an Fab, Fab′, Fv, F(ab′)₂ or an antigen binding scaffold(e.g., affibody, monobody, anticalin, DARPin, Knottin).

the terms “binds,” “binding” or grammatical equivalents, refer tocompositions, directly or indirectly, having affinity for each other.“Specific binding” is where the binding is selective between twomolecules. A particular example of specific binding occurs between anantibody and an antigen. Typically, specific binding can bedistinguished from non-specific when the dissociation constant (K_(D))is less than about 1×10⁻⁵M or less than about 1×10⁻⁶ M or 1×10⁻⁷M.Specific binding can be detected, for example, by ELISA,immunoprecipitation, coprecipitation, with or without chemicalcrosslinking, and two-hybrid assays. Appropriate controls can be used todistinguish between “specific” and “non-specific” binding. “Affinity” isthe strength of the binding interaction of two molecules, such as anantigen and its antibody, which is defined for antibodies and othermolecules with more than one binding site as the strength of binding ofthe ligand at one specified binding site. Although the noncovalentattachment of a ligand to antibody or other molecule is typically not asstrong as a covalent attachment, “high affinity” is for a ligand thatbinds to an antibody or other molecule having an affinity constant(K_(a)) of greater than 10⁴ M⁻¹, typically 10⁵-10¹¹ M⁻¹; as determinedby inhibition ELISA or an equivalent affinity determined by comparabletechniques, such as Scatchard plots or using K_(J)/dissociationconstant, which is the reciprocal of the K_(a).

The term “selective” with respect to binding, inhibition, stimulation,or modulation means preferential binding, inhibition, stimulation, ormodulation, respectively, of a first activity relative to a secondactivity (e.g., preferential binding of one receptor to anotherreceptor; preferential inhibition relative to other receptors; orpreferential inhibition of a mutant to a wild-type or vice versa). Insome cases, binding is greater than two times more selective, greaterthan five times more selective, greater than ten times more selective,greater than fifty times more selective, greater than 100 times moreselective, or greater than 1000 times more selective for the desiredmolecular target or pathway versus an undesired molecular target orpathway. In some cases, a compound will bind a first molecular target oraffect a pathway by at least 2-fold, at least 5-fold, at least 10-fold,at least 20-fold, at least 50-fold, at least 100-fold relative to asecond target or pathway under the same conditions. It will beappreciated that in preferred embodiments, binding to the D2-like familyof dopamine receptors or a member thereof, will be selective withrespect to the D1-like family of dopamine receptors or a member thereofby any of the foregoing amounts. The in vitro or in vivo activity of amolecular target or pathway may be measured by any suitable reproduciblemeans.

The term “modulating” refers to “stimulating” or “inhibiting” anactivity of a molecular target or pathway. For example, a compositionmodulates the activity of a molecular target or pathway if it stimulatesor inhibits the activity of that target or pathway by at least 10%, byat least about 20%, by at least about 25%, by at least about 30%, by atleast about 40%, by at least about 50%, by at least about 60%, by atleast about 70%, by at least about 75%, by at least about 80%, by atleast about 90%, by at least about 95%, by at least about 98%, or byabout 99% or more relative to the activity of that molecular target orpathway under the same conditions but lacking only the presence of thecomposition. In another example, a composition modulates the activity ofa molecular target or pathway if it stimulates or inhibits the activityof that target or pathway by at least 2-fold, at least 5-fold, at least10-fold, at least 20-fold, at least 50-fold, at least 100-fold relativeto the activity of that target or pathway under the same conditions butlacking only the presence of the composition. The activity of amolecular target or pathway may be measured by any reproducible means.For example, the activity of a molecular target or pathway may bemeasured in vitro or in vivo by a suitable assay known in the art formeasuring the activity. Control samples (untreated with the composition)can be assigned a relative activity value of 100%.

In one embodiment, an antibody, antigen-binding fragment, or affinitytag binds its target with a K_(D) of 0.1 nM-10 mM, 0.1 nM-1 mM, orwithin the 0.1 nM range. In one embodiment, an antibody, antigen-bindingfragment, or affinity tag binds its target with a K_(D) of 0.1-2 nM,0.1-1 nM, 0.05-1 nM, 0.1-0.5 nM, or 0.1-0.2 nM. In one embodiment, anantibody, antigen-binding fragment, or affinity tag bind its targetdirectly. In one embodiment, an antibody, antigen-binding fragment, oraffinity tag bind its target indirectly, for example, binding as asecondary antibody that binds to an antibody bound to the target.

The word “label” refers to a compound or composition which is conjugatedor fused directly or indirectly to a reagent such as a nucleic acidprobe or an antibody and facilitates detection of the reagent to whichit is conjugated or fused. The label may itself be detectable (e.g.,radioisotope or fluorescent labels) or, in the case of an enzymaticlabel, may catalyze chemical alteration of a substrate compound orcomposition, which is detectable.

The term “probe” refers to synthetic or biologically produced nucleicacids that contain specific nucleotide sequences which hybridize understringent conditions to target nucleic acid sequences. The terms“labeled probe,” “nucleic acid probe operably linked to a detectablelabel,” or “nucleic acid strand operably linked to a detectable label”refer to a probe which is prepared with a marker moiety or “detectablelabel” for detection. The marker moiety is attached at either the 5′end, the 3′ end, internally, or a combination thereof. That is, oneprobe may be attached to multiple marker moieties. A preferred moiety isan identifying label such as a fluorophore. A labeled probe may alsocomprise a plurality of different nucleic acid sequences each labeledwith one or more marker moieties. Each marker moiety may be the same ordifferent. It may be beneficial to label different probes (e.g., nucleicacid sequences) each with a different marker moiety. This can beachieved by having a single distinguishable moiety on each probe. Forexample, probe A is attached to moiety X and probe B is attached tomoiety Y. Alternatively, probe A is attached to moieties X and Y whileprobe B is attached to moiety Z and W. Alternatively, probe A isattached to moieties X and Y, while probe B is attached to moieties Yand Z. All probes “A” and “B” above would be distinguishable anduniquely labeled.

By “tissue sample” is meant a collection of similar cells obtained froma tissue of a subject or patient, preferably containing nucleated cellswith chromosomal material. The four main human tissues are (1)epithelium; (2) connective tissues, including blood vessels, bone andcartilage; (3) muscle tissue; and (4) nerve tissue. The tissue samplesource may be solid tissue as from a fresh, frozen and/or preservedorgan or tissue sample or biopsy or aspirate; blood or a bloodconstituent; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid, or interstitial fluid; cells from a time ingestation or development of the subject. A tissue sample may be primaryor cultured cells or cell lines. A tissue sample may contain compoundsthat are not naturally intermixed with the tissue in nature such aspreservatives, anticoagulants, buffers, fixatives, nutrients, orantibiotics. By a “section” of a tissue sample is meant a single part orpiece of a tissue sample, e.g., a thin slice of tissue or cells cut froma tissue sample. Multiple sections of tissue samples may be taken andsubjected to analysis. A “cell line” refers to a permanently establishedcell culture that will proliferate given appropriate fresh medium andspace.

Detection Methods

In various aspects, provided herein are methods of detecting ormeasuring a target receptor (e.g., a dopamine receptor or a GPCR) in abiological sample. Targets are detected by contacting the sample with atarget detection reagent, e.g., an antibody or fragment thereof, and alabeling reagent. The presence or absence of targets are detected by thepresence or absence of the labeling reagent. In some instances, a sampleis contacted with the target detection and the labeling reagentsconcurrently e.g., the detection reagent is a primary antibody and thelabeling reagent is a fluorescent dye conjugated to it. Alternatively,the biological sample is contacted with the target detection andlabeling reagents sequentially, e.g., the detection reagent is a primaryantibody and the labeling reagent includes a secondary antibody. Forexample, a sample is incubated with a detection reagent, in some casestogether with a labeling reagent, under conditions that allow a complexbetween the detection reagent (and labeling reagent) and target to form.After complex formation the sample is optionally washed one or moretimes to remove unbound detection reagent (and labeling reagent). Whenthe sample is further contacted with a labeling reagent thatspecifically binds the detection reagent bound to the target, the samplecan optionally be washed one or more times to remove unbound labelingreagent. The presence or absence of the target in the sample is thendetermined by detecting the labeling reagent.

The methods described here provide for detection of multiple targets ina sample. Multiple targets are identified by contacting the biologicalsample with additional detection reagents followed by additionallabeling reagent specific for the additional detection reagents usingthe methods described.

A detection moiety, i.e., detectable label, is a substance used tofacilitate identification and/or quantitation of a target. Detectionmoieties are directly observed or measured or indirectly observed ormeasured. Detection moieties include, but are not limited to,radiolabels that can be measured with radiation-counting devices;pigments, dyes or other chromogens that can be visually observed ormeasured with a spectrophotometer; spin labels that can be measured witha spin label analyzer; and fluorescent moieties, where the output signalis generated by the excitation of a suitable molecular adduct and thatcan be visualized by excitation with light that is absorbed by the dyeor can be measured with standard fluorometers or imaging systems. Thedetection moiety can be a luminescent substance such as a phosphor orfluorogen; a bioluminescent substance; a chemiluminescent substance,where the output signal is generated by chemical modification of thesignal compound; a metal-containing substance; or an enzyme, where anenzyme-dependent secondary generation of signal occurs, such as theformation of a colored product from a colorless substrate. The detectionmoiety may also take the form of a chemical or biochemical, or an inertparticle, including colloidal gold, microspheres, quantum dots, orinorganic crystals such as nanocrystals or phosphors. The term detectionmoiety or detectable label can also refer to a “tag” or hapten that canbind selectively to a labeled molecule such that the labeled molecule,when added subsequently, is used to generate a detectable signal. Forinstance, one can use biotin, iminobiotin or desthiobiotin as a tag andthen use an avidin or streptavidin conjugate of horseradish peroxidase(HRP) to bind to the tag, and then use a chromogenic substrate (e.g.,tetramethylbenzidine) or a fluorogenic substrate such as Amplex Red orAmplex Gold (Molecular Probes, Inc.) to detect the presence of HRPSimilarly, the tag can be a hapten or antigen (e.g., digoxigenin), andan enzymatically, fluorescently, or radioactively labeled antibody canbe used to bind to the tag. Numerous labels are known by those of skillin the art and include, but are not limited to, particles, fluorescentdyes, haptens, enzymes and their chromogenic, fluorogenic, andchemiluminescent substrates.

A fluorophore is a chemical moiety that exhibits an absorption maximumbeyond 280 nm, and when covalently attached in a labeling reagentretains its spectral properties. Fluorophores include a pyrene, ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzoindole, an oxazole or benzoxazole, a thiazole or benzothiazole, aporphyrin, a cyanine, a perylene, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a carbocyanine, acarbostyryl, a salicylate, an anthranilate, an azulene, a pyridine, aquinoline, a borapolyazaindacene, a xanthene, an oxazine or abenzoxazine, a carbazine, a phenalenone, a coumarin, a benzofuran andbenzphenalenone and derivatives thereof. oxazines include resorufins,aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

When the fluorophore is a xanthene, the fluorophore may be afluorescein, a rhodol, or a rhodamine. Fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, rhodol includes seminaphthorhodafluors. Alternatively, thefluorophore is a xanthene that is bound via a single covalent bond atthe 9-position of the xanthene. Preferred xanthenes include derivativesof 3H-xanthen-6-ol-3-one, derivatives of 6-amino-3H-xanthen-3-one, orderivatives of 6-amino-3H-xanthen-3-imine Fluorophores include xanthene(rhodol, rhodamine, fluorescein and derivatives thereof) coumarin,cyanine, pyrene, oxazine and borapolyazaindacene. In addition, thefluorophore can be sulfonated xanthenes, fluorinated xanthenes,sulfonated coumarins, fluorinated coumarins and sulfonated cyanines. Thechoice of fluorophore in the labeling reagent will determine theabsorption and fluorescence emission properties of the labeling reagent.Physical properties of a fluorophore label include spectralcharacteristics (absorption, emission and stokes shift), fluorescenceintensity, lifetime, polarization and photo-bleaching rate can all beused to distinguish one fluorophore from another.

Typically, a fluorophore contains one or more aromatic or heteroaromaticrings that are optionally substituted by one or more of a variety ofsubstituents, including halogen, nitro, cyano, alkyl, perfluoroalkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl orheteroaryl ring system, benzo, or other substituents typically presenton fluorophores known in the art.

Preferably, the detection moiety is a fluorescent dye. Fluorescent dyesinclude, for example, Fluorescein, Rhodamine, Texas Red, Cy2, Cy3, Cy5,Cy0, Cy0.5, Cy1, Cy1.5, Cy3.5, Cy7, VECTOR Red, ELF™ (Enzyme-LabeledFluorescence), FluorX, Calcein, Calcein-AM, CRYPTOFLUOR™'S, Orange (42kDa), Tangerine (35 kDa), Gold (31 kDa), Red (42 kDa), Crimson (40 kDa),BUMP, BHDMAP, Br-Oregon, Lucifer Yellow, Alexa dye family,N-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl) (NBD), BODIPY™,boron dipyrromethene difluoride, Oregon Green, MITOTRACKER™ Red, DiOC7(3), DiIC18, Phycoerythrin, Phycobiliproteins BPE (240 kDa) RPE (240kDa) CPC (264 kDa) APC (104 kDa), Spectrum Blue, Spectrum Aqua, SpectrumGreen, Spectrum Gold, Spectrum Orange, Spectrum Red, NADH, NADPH, FAD,Infra-Red (IR) Dyes, Cyclic GDP-Ribose (cGDPR), Calcofluor White,Tyrosine and Tryptophan. Many fluorophores can also function aschromophores and thus they are also preferred chromophores.

In addition to fluorophores, enzymes also find use as detectablemoieties. Enzymes are desirable detectable moieties becauseamplification of a detectable signal can be achieved resulting inincreased assay sensitivity. The enzyme itself does not produce adetectable response but breaks down a substrate when it is contacted byan appropriate substrate such that the converted substrate produces afluorescent, colorimetric or luminescent signal. Enzymes amplify adetectable signal because one enzyme on a labeling reagent can result inmultiple substrates being converted to a detectable signal. This isadvantageous where there is a low quantity of target present in thesample or a fluorophore does not exist that will give comparable orstronger signal than the enzyme. However, fluorophores are preferredbecause they do not require additional assay steps, and thus reduce theoverall time to complete an assay. The enzyme substrate is selected toyield the preferred measurable product, e.g. colorimetric, fluorescentor chemiluminescence. Such substrates are extensively used in the art.

A preferred colorimetric or fluorogenic substrate and enzyme combinationuses oxidoreductases such as horseradish peroxidase and a substrate suchas 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazol-e (AEC),which yield a distinguishing color (brown and red, respectively). Othercolorimetric oxidoreductase substrates that yield detectable productsinclude, but are not limited to:2,2-azino-bis(3-ethylbenzothiaz-oline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates include, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplexe Red reagent and its variants andreduced dihydroxanthenes, including dihydrofluoresceins anddihydrorhodamines including dihydrorhodamine 123. Peroxidase substratesthat are tyramides represent a unique class of peroxidase substrates inthat they can be intrinsically detectable before action of the enzymebut are “fixed in place” by the action of a peroxidase in the processdescribed as tyramide signal amplification (TSA). These substrates areextensively utilized to label targets in samples that are cells, tissuesor arrays for their subsequent detection by microscopy, flow cytometry,optical scanning and fluorometry.

Additional colorimetric (and in some cases fluorogenic) substrate andenzyme combination use a phosphatase enzyme such as an acid phosphatase,an alkaline phosphatase or a recombinant version of such a phosphatasein combination with a colorimetric substrate such as5-bromo-6-chloro-3-indolyl phosphate (BOP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy4-methylcoumarinyl phosphate (DiFMUP) fluoresceindiphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAOphosphate), or ELF 97, ELF 39 or related phosphates.

Glycosidases, in particular β-galactosidase, β-glucuronidase andβ-glucosidase, are additional suitable enzymes. Appropriate colorimetricsubstrates include, but are not limited to, 5-bromo4-chloro-3-indolylβ-D-galactopyranoside (X-gal) and similar indolyl galactosides,glucosides, and glucuronides, o-nitrophenyl β-D-galactopyranoside (ONPG)and p-nitrophenyl β-D-galactopyranoside. Preferred fluorogenicsubstrates include resorufin β-D-galactopyranoside, fluoresceindigalactoside (FDG), fluorescein diglucuronide and their structuralvariants, 4-methylumbelliferyl β-D-galactopyranoside,carboxyumbelliferyl β-D-galactopyranoside and fluorinated coumarinβ-D-galactopyranosides. Additional enzymes include hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases, and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare preferred for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful. For example,the enzyme is luciferase or aequorin. The substrates are luciferine,ATP, Ca⁺⁺ and coelenterazine.

In addition to enzymes, haptens such as biotin are useful detectablemoieties. Biotin is useful as it is in an enzyme system that can furtheramplify a detectable signal, and it can serve as a tag in affinitychromatography for isolation purposes. For detection, an enzymeconjugate that has affinity for biotin is used, such as avidin-HRP.Subsequently, a peroxidase substrate is added to produce a detectablesignal. Haptens also include hormones, naturally occurring and syntheticdrugs, pollutants, allergens, affector molecules, growth factors,chemokines, cytokines, lymphokines, amino acids, peptides, chemicalintermediates, or nucleotides.

In some cases, a detectable moiety is a fluorescent protein. Exemplaryfluorescent proteins include green fluorescent protein (GFP),phycobiliproteins and their derivatives, luciferase or aequorin.Fluorescent proteins, especially phycobiliprotein, are particularlyuseful for creating tandem dye labeled labeling reagents. These tandemdyes comprise a fluorescent protein and a fluorophore to obtain a largerstokes shift where the emission spectra is farther shifted from thefluorescent protein's absorption spectra. This is particularlyadvantageous to detect a low amount of target in a sample where theemitted fluorescent light is maximally optimized, in other words thefluorescent protein reabsorbs little to none of the emitted light. Thefluorescent protein and fluorophore function as an energy transfer pairwhere the fluorescent protein emits at a wavelength the fluorophoreabsorbs, and the fluorophore then emits at a wavelength farther from thefluorescent protein than could be obtained with only the fluorescentprotein. A particularly useful combination is phycobiliproteins andsulforhodamine fluorophores, or sulfonated cyanine fluorophores; orsulfonated xanthene derivatives. Alternatively, the fluorophore is anenergy donor and the fluorescent protein is an energy acceptor.

Methods of Visualizing the Detection Moiety Depend on the Label.

In some cases, a sample is illuminated with a light wavelength selectedto give a detectable optical response, and observed with a means fordetecting that response. Equipment useful for illuminating fluorescentcompounds include hand-held ultraviolet lamps, mercury arc lamps, xenonlamps, lasers and laser diodes. These illumination sources are opticallyintegrated into laser scanners, fluorescent microplate readers orstandard or microfluorometers. The degree or location of signal,compared to a standard or expected response, indicates whether and towhat degree the sample possesses a given characteristic or desiredtarget.

An optical response is detected by visual inspection, or by using one ofthe following devices: CCD camera, video camera, photographic film,laser-scanning devices, fluorometers, photodiodes, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or by means for amplifying the signalsuch as photomultiplier tubes. When a sample is examined using a flowcytometer, examination of it optionally includes sorting portions of itaccording to their fluorescence response.

When an indirectly detectable label is used, then illuminating typicallyincludes adding a reagent to produce a detectable signal such as acolorimetric enzyme substrate. Radioisotopes are also consideredindirectly detectable where an additional reagent is not needed, ratherthe radioisotope is exposed to X-ray film or other mechanism to recordand measure the signal. This is true for some chemiluminescent signalsthat are observed after exposure to film.

I. ONC201 (COMPOUND (1)), SALTS THEREOF AND SYNTHESES THEREOF

ONC201 (compound (1))

and its analogs, and their pharmaceutically acceptable salts, as well assyntheses for them, are provided herein. In in vitro models, animalmodels, and human clinical trials, ONC201 has broad anti-canceractivity, low toxicity including few, if any, adverse effects, lowgenotoxicity, and high bioavailability including orally. These featuresallow ONC201 and various analogs to be well suited for a variety ofapplications. ONC201 can be made by the synthesis shown in Scheme 1.

Synthesis of an ONC201 dihydrochloride salt starts with commerciallyavailable intermediary N-Benzyl-3-carbomethoxy-4-piperidonehydrochloride, compound (3). In one embodiment, the synthesis includesneutralizing compound (3) with a base (Step 1) to produce compound (4),a free base. In one embodiment, compound (3) is neutralized with aninorganic base to produce compound (4). In one embodiment, compound (3)is neutralized with an organic base to produce compound (4). In oneembodiment, compound (3) is neutralized in the presence of an alcohol,for example, n-butanol. In one embodiment, compound (3) is neutralizedin the presence of at least one organic solvent, for example, n-butanoland/or ethyl acetate. In one embodiment, compound (3) is neutralized inthe presence of a base and at least one organic solvent, for example,NaHCO₃ and n-butanol. In one embodiment, compound (3) is neutralized inthe presence of n-butanol and triethyl amine (Et₃N).

In one embodiment, the synthesis includes reacting compound (4) withcompound (5) (Step 2) to produce intermediary compound (1). In oneembodiment, the reaction in Step 2 includes heating compound (4) withcompound (5). In one embodiment, the reaction in Step 2 includesrefluxing heating compound (4) and compound (5) in the presence of asolvent. In one embodiment, the reaction in Step 2 includes use ofDean-stark trap to remove water and/or methanol (MeOH) formed in thereaction.

In one embodiment, an ONC201 dihydrochloride salt is synthesized (Step3). In one embodiment, this reaction (Step 3) includes treating ONC201with HCl in dioxane. In one embodiment, Step 3 includes treating ONC201with 4N HCl in dioxane. In one embodiment, the synthesis optionallyincludes recrystallizing the ONC201 di-salt. In a preferred embodiment,the ONC201 di-hydrochloride salt is synthesized as shown in Scheme 2.

II. TNF-RELATED APOPTOSIS-INDUCING LIGAND (“TRAIL”)

TRAIL protein can be assayed in a sample obtained from a subject todetect TRAIL expression induced by compounds and their salts describedherein Immunoassays can be used to assay TRAIL in a sample, includingenzyme-linked immunosorbent assay (ELISA), enzyme-linkedimmunofiltration assay (ELIFA), flow cytometry, immunoblot,immunoprecipitation, immunohistochemistry, immunocytochemistry,luminescent immunoassay (LIA), fluorescent immunoassay (FIA), andradioimmunoassay. Assays may be used to obtain qualitative and/orquantitative results. Specific details of suitable methods for bothqualitative and quantitative sample assays are described in standardreferences, including E. Harlow & D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling & S.Diibel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H.Zola, Monoclonal Antibodies: Preparation and Use of MonoclonalAntibodies and Engineered Antibody Derivatives, Basics: From Backgroundto Bench, BIOS Scientific Publishers, 2000; B. K. C. Lo, AntibodyEngineering: Methods and Protocols, Methods in Molecular Biology, HumanaPress, 2003; F. M. Ausubel et al., Eds., Short Protocols in MolecularBiology, Current Protocols, Wiley, 2002; S. Klussman, Ed., The AptamerHandbook: Functional Oligonucleotides and Their Applications, Wiley,2006; Ormerod, M. G., Flow Cytometry: a practical approach, OxfordUniversity Press, 2000; Givan, A. L., Flow Cytometry: first principles,Wiley, New York, 2001; Gorczyca, W., Flow Cytometry in NeoplasticHematology: morphologic-immunophenotypic correlation, Taylor & Francis,2006; Crowther, J. R., The ELISA Guidebook (Methods in MolecularBiology), Humana Press, 2000; Wild, D., The Immunoassay Handbook, 3^(rd)Edition, Elsevier Science, 2005, and J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 3^(rd) ed., 2001.

Protocols to assay and analyze a sample for TRAIL to detect an effect ofa pharmaceutical composition are described in U.S. Pat. No. 8,673,923 toWafik S. El-deiry et al., which is incorporated by reference herein inits entirety.

In one embodiment, TRAIL assays are used to monitor a subject. Forexample, a sample is obtained from a subject before treatment with apharmaceutical composition and at one or more times during and/orfollowing treatment to assess the treatment's effectiveness. In anotherexample, a sample is obtained from a subject at various times to assessthe course or progress of disease or healing. In one embodiment, deathreceptors from circulating tumor cells are assayed to see if a treatmentdescribed here increases the amount or type of death receptors.

Cancers treated using methods and compositions described herein arecharacterized by abnormal cell proliferation including pre-neoplastichyperproliferation, cancer in-situ, neoplasms and metastasis. Methodsand compositions described herein can be used for prophylaxis, as wellas amelioration of cancer signs or symptoms. “Treatment” of a cancer ina subject includes: preventing, inhibiting or ameliorating cancer in thesubject, such as slowing cancer progression or reducing or amelioratinga cancer sign or symptom. Examples of cancers treated using methods andcompositions described herein include breast cancer, CNS cancers, coloncancer, ovarian cancer, prostate cancer, leukemia, lung cancer, andlymphoma.

III. COMPOUNDS OF FORMULA (10) AND SALTS THEREOF

In one aspect, provided herein are compounds and salts of formula (10)and methods of making them. Persons skilled in the art will understandthat the general principles and concepts described here in conjunctionwith ONC201 (compound (1)) and its salts, including principles andconcepts related to methods and pharmaceutical compositions, apply withequal force to compounds of formula (10) and salts thereof.

In one embodiment, provided herein are compounds of formula (10):

wherein R₁ and R₂ are independently selected from H, alkyl, aryl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl,aralkoxy, aralkylthio, and acyl radicals. In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph) (i.e., ONC201). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2,4-di F-Ph) (i.e., ONC206). In one embodiment, R₁ isCH₂Ph and R₂ is CH₂-(4-CF₃-Ph) (i.e., ONC212). In one embodiment, R₁ isCH₂Ph and R₂ is CH₂-(3,4-di F-Ph) (i.e., ONC213). In one embodiment, R₁is CH₂ (3,4-di-Cl-Ph and R₂ is CH₂-(4-CF₃-Ph) (i.e., ONC234). In oneembodiment, R₁ is CH₂-3-thienyl and R₂ is CH₂-(4-CF₃-Ph) (i.e., ONC236).

In one embodiment, R₁ and R₂ are independently selected from the groupconsisting of H, C₁₋₄ alkyl, C₁₋₄ alkylphenyl, C₁₋₄ alkylphenylketone,C₁₋₄ benzyl-piperazine, C₁₋₄alkylthienyl, C₁₋₄alkylpyridinyl,C₁₋₄alkylisoxazolidinyl, C₁₋₄alkylmorpholinyl, C₁₋₄alkylthiazolyl, andC₁₋₄alkylpyrazinyl wherein C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄ benzyl-piperazine, C₁₋₄alkylthienyl,C₁₋₄alkylpyridinyl, C₁₋₄ alkylisoxazolidinyl, C₁₋₄ alkylmorpholinyl,C₁₋₄alkylthiazolyl, and C₁₋₄alkylpyrazinyl are optionally substitutedwith C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, orhalo. In one embodiment, R₁ and/or R₂ is a substituted or unsubstituted,arylalkyl or heteroarylalkyl. In one embodiment, the heteroarylalkyl isselected from C₁₋₄alkylpyrrolyl, C₁₋₄alkylfuryl, C₁₋₄alkylpyridyl,C₁₋₄alkyl-1,2,4-thiadiazolyl, C₁₋₄alkylpyrimidyl, C₁₋₄alkylthienyl, C₁₋₄alkylisothiazolyl, C₁₋₄ alkylimidazolyl, C₁₋₄alkyltetrazolyl,C₁₋₄alkylpyrazinyl, C₁₋₄ alkylpyrimidyl, C₁₋₄ alkylquinolyl,C₁₋₄alkylisoquinolyl, C₁₋₄alkylthiophenyl, C₁₋₄ alkylbenzothienyl, C₁₋₄alkylisobenzofuryl, C₁₋₄alkylpyrazolyl, C₁₋₄alkylindolyl, C₁₋₄alkylpurinyl, C₁₋₄ alkylcarbazolyl, C₁₋₄ alkylbenzimidazolyl, and C₁₋₄alkylisoxazolyl.

In one embodiment, R₁ and/or R₂ is a benzyl optionally substituted withone or more of the following substituents on the benzyl ring: X, —CH₃,—NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂-C₄ alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1), —OC_(p)X_(2p+1),OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m),C(O)R_(m), and C(O)OR^(m); R^(m) and R^(n) are independently selectedfrom H or a C₁-C₄ alkyl; and where p is an integer from 2 to 20 and X isa halogen, including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl.

In one embodiment, R₁ is selected from H, CH₃, CH₂Ph, CH₂-(4-CF₃-Ph),CH₂-(4-F-Ph), CH₂-(4-Cl-Ph), CH₂—(OCH₃-Ph), CH₂-((2-Cl)-Ph),CH₂-(2-thienyl), CH₂-(3-thienyl), CH₂-2-pyridinyl,CH₂-4-methyl-2-thiazolyl, CH₂-2-pyrazinyl, CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-(3,4-di Cl-Ph),CH₂-(3,4-di F-Ph), CH₂-(3,5-di F-Ph), CH₂-((2-CH₃)-Ph), CH₂CH(OH)Ph,(4-F-Ph)-4-oxobutyl, CH₂CH₂NHCOOC(CH₃)₃, CH₂CH₂CH₂NH₂, and CD₂C₆D₅. Inone embodiment, R₂ is selected from H, CH₃, CH₂Ph, CH₂-(4-CF₃-Ph),CH₂-((2-Cl)-Ph), CH₂-((2-F)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-(2,4-di Cl-Ph),CH₂-(3,4-di Cl-Ph), CH₂-(3,4-di F-Ph), CH₂-(3,5-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂(2-CH₃, 4-F-Ph), CH₂-((4-OCH₃)-Ph),CH₂-(3-pyridinyl), CH₂-(3-isoxazolidinyl), CH₂CH₂-(4-morpholinyl),CH₂-(2-F, 4-CF₃-Ph), CH₂CH(OH)Ph, (CH₂)₃CO-4F-Ph, (4-F-Ph)-4-oxobutyl,CH₂CH₂NHCOOC(CH₃)₃, CH₂CH₂CH₂NH₂, and CD₂C₆D₅.

In one embodiment, R₁ is H. In one embodiment, R₁ is a substituted orunsubstituted arylalkyl, e.g., a benzyl (CH₂Ph) or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,e.g., benzyl or phenylethyl. In one embodiment, the arylalkyl issubstituted with C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from halo, CH₃, CF₃ or OCH₃. In oneembodiment, R₂ is a substituted or an unsubstitutedheterocycloalkylalkyl, e.g., piperazinylalkyl or morpholinoalkyl. In oneembodiment, R₂ is a substituted or an unsubstituted heteroarylalkyl,e.g., pyridylmethyl or isoxazolidinylmethyl. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with C₁₋₄ alkyl,C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, or halo. In oneembodiment, the heterocycloalkylalkyl or heteroarylalkyl is substitutedwith at least one substituent selected from halo, CH₃, CF₃ or OCH₃.

In one embodiment, compound (10) has the structure of formula (80):

wherein R_(a1), R_(a2), R_(a3), R_(a4), R_(a5), R_(b1), R_(b2), R_(b3),R_(b4), and R_(b5) are each independently selected from the groupconsisting of H, X, —CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂-C₄ alkyl,—CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃,—OC_(p)H_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n),NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) andR^(n) are independently selected from H or a C₁-C₄ alkyl; and where p isan integer from 2 to 20 and X is a halogen.

In one embodiment, compound (10) has the structure of formula (90)

wherein R₂ is as defined above, and wherein R_(b1), R_(b2), R_(b3),R_(b4), and R_(b5) are each independently selected from the groupconsisting of H, X, —CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂₋₄ alkyl,—CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃,—OC_(p)H_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n),NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) andR^(n) are independently selected from H or a C₁₋₄alkyl; and where p isan integer from 2 to 20 and X is a halogen.

In one embodiment, compound (10) has the structure of formula (40)

where R₁ is as defined above, and where R_(a1), R_(a2), R_(a3), R_(a4),and R_(a5) are each independently selected from the group consisting ofH, X, —CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂₋₄ alkyl, —CX₃, —CH₂(CX₃),—CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1),—OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m),SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) and R^(n) are independentlyselected from H or a C₁₋₄ alkyl; p is an integer from 2 to 20; and X isa halogen. In one embodiment, R₁ is H. In one embodiment, R₁ is asubstituted or unsubstituted arylalkyl, such as benzyl or phenylethyl.In one embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo. In oneembodiment, the benzyl is substituted with one or more halo. In oneembodiment, the benzyl is substituted with one or more substituentsselected from halo, CH₃, CF₃, and OCH₃. In one embodiment, the benzyl issubstituted with one halo, e.g., F at an ortho or para position. In oneembodiment, the benzyl is substituted with two halogen, e.g., F at bothmeta positions.

In one embodiment, compound (40) has the structure of compound (45):

where R_(a1), R_(a2), R_(a3), R_(a4), and R_(a5) are as defined above.In one embodiment, the benzyl is substituted with one or more halogens.In one embodiment, the benzyl is substituted with one or moresubstituents selected from halo, CH₃, CF₃, and OCH₃. In one embodiment,R_(a1) or R_(a5) is a halo, e.g., F. In one embodiment, both R_(a2) andR_(a3) are halo, e.g., F.

In one embodiment, compound (10) has the structure of compound (50)

wherein R₁ is as defined above, and wherein R_(b) is selected from thegroup consisting of H, X, —CH₃, —NO₂, —OCH₃, —CN, C₂₋₄ alkyl, —CX₃,—CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1),—OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m),SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) and R^(n) are independentlyselected from H or C₁₋₄alkyl; and where p is an integer from 2 to 20 andX is a halogen, and wherein R_(a1), R_(a2), R_(a4), and R_(a)s are eachindependently selected from the group consisting of H, X, —CH₃, —NO₂,—OCH₃, —CN, —CXH₂—CX₂H C₂₋₄ alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, —OC_(p)X_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m),NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m), C(O)R^(m), andC(O)OR^(m); R^(m) and R^(n) are independently selected from H or C₁₋₄alkyl; and where p is an integer from 2 to 20 and X is a halogen. In oneembodiment, R₁ is H. In one embodiment, R₁ is a substituted orunsubstituted arylalkyl, such as a benzyl or phenylethyl group. In oneembodiment, the arylalkyl is substituted with C₁₋₄alkyl, C₁₋₄ alkoxyl,hydroxyl, perhalogenated C₁₋₄ alkyl, or halo. In one embodiment, R_(b)is selected from halo, CH₃, CF₃, and OCH₃. In one embodiment, one ormore of R_(a1), R_(a2), R_(a4), and R_(a5) is selected from halo, CH₃,CF₃, and OCH₃. In one embodiment, R_(a1), R_(a2), R_(a4), and R_(a5) areH, and R_(b) is selected from halo, CH₃, CF₃, and OCH₃. In oneembodiment, R_(b) is a halogen, e.g., F, and R_(a1) is CH₃. In oneembodiment, R_(b) is F or Cl, and R_(a2) is F or Cl. In one embodiment,R_(b) is CF₃. In one embodiment, R_(b) is OCH₃. In one embodiment, R_(b)and R_(a1) are Cl.

In one embodiment, compound (50) has the structure of compound (55):

where R_(a1), R_(a2), R_(a4), R_(a5), and R_(b) are as defined above. Inone embodiment, R_(b) is selected from halo, CH₃, CF₃, and OCH₃. In oneembodiment, one or more of R_(a1), R_(a2), R_(a4), and R_(a5) isselected from halo, CH₃, CF₃, and OCH₃. In one embodiment, R_(a1),R_(a2), R_(a4), and R_(a5) are H, and R_(b) is selected from halo, CH₃,CF₃, and OCH₃. In one embodiment, R_(b) is halo, e.g., F, and R_(a1) isCH₃. In one embodiment, R_(b) is F or Cl, and R_(a2) is F or Cl. In oneembodiment, R_(b) is CF₃. In one embodiment, R_(b) is OCH₃. In oneembodiment, R_(b) and R_(a1) are Cl.

In one embodiment, compound (10) has the structure of compound (60)

In one embodiment, R₁ is H. In one embodiment, R₁ is a substituted orunsubstituted arylalkyl, such as benzyl or phenylethyl. In oneembodiment, R₁ is a substituted or unsubstituted heterocycloalkylalkylor a substituted or unsubstituted heteroarylalkyl, such asCH₂-(2-thienyl), CH₂-(3-thienyl), CH₂-4-methyl-2-thiazolyl,CH₂-2-pyrazinyl, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(3-isoxazolidinyl),CH₂-2-pyridinyl, CH₂-3-pyridinyl, and CH₂CH₂-(4-morpholinyl). In oneembodiment, the arylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl,hydroxyl, perhalogenated C₁₋₄alkyl, or halo. In one embodiment, thebenzyl is substituted with one or more halogens. In one embodiment, thebenzyl is substituted with one or more substituents selected from halo(e.g., F), CH₃, CF₃, and OCH₃. In one embodiment, the benzyl issubstituted at the para position with a halo, CH₃, CF₃, or OCH₃substituent. In one embodiment, R₁ is fluorophenyloxobutyl orhydroxyphenylethyl Scheme 3 illustrates the synthesis of compounds offormula (10):

Compounds of formula (10) (i.e., imipridones) are synthesized startingfrom a substituted piperidone, which is converted by reaction with asubstituted aminoimidazoline to give the core compound (10). There aretwo routes, one in which the R₁ substituent is present in the piperidone(e.g., 68). In that route, (68) is acylated with dimethyl carbonateusing sodium hydride in toluene at 80° C. to form piperidone ester (69).Commercially available methylthioimidazoline HI salt (63) is reactedwith an amine in dioxane at 70° C. to afford the R₂-substitutedaminoimidazoline (64) as its HI salt. Direct reaction of (64) withpiperidone ester (69) in 1-butanol at reflux with removal of water via aDean-Stark trap over 3-6 h gives the tricyclic compound (10). In avariant of this scheme, N—BOC protected piperidone (61) is converted bythe same methods to BOC protected compound (65), which is treated withHCl in dioxane to remove the BOC group and then converted to the freebase of (66) with 1N NaOH with extraction with methylene chloride.Subsequent treatment of (66) with a halide (67) or epoxide (70) affordsdesired compound (10).

Crude products may be purified by column chromatography eluting withmethylene chloride:methanol or by HPLC using acetonitrile:TFA:H₂O toproduce final products as either free bases or as TFA salts. Treatmentof free bases with HCl in dioxane or lyophilization of TFA saltsgenerates products (10) as HCl or TFA salts. Alternatively, the freebase may be treated with another inorganic or organic acid to form othersalts, generally selected from those known to be pharmaceuticallyacceptable. Salts of compound (10) are usually solids and examples havebeen crystallized from ethanol or other solvents to give high qualitycrystals. The tricyclic structure has been definitively confirmed in thecase of compound (1) by an X-ray crystal structure and NMR.

Compounds described herein can be used, with or without an aminoalkyllinker (e.g., compound (33)), to identify molecules (e.g., proteins)that interact with them in a cellular context. Expression of thesebinding targets may be used to predict response to imipridones oranalogs thereof (i.e. serve as biomarkers). These compounds can also beused to screen for structurally unrelated molecules using competitionassays known in the art to identify drugs able to outcompete the targetinteraction with a higher affinity. In addition, these molecules mayhave improved drug properties or allow additional applications byaltering drug properties including safety, potency, pharmacokinetics,biodistribution, or metabolism.

TABLE 1 EXAMPLES OF COMPOUNDS OF FORMULA (10) No. ONC Number R₁ R₂  1ONC201 CH₂Ph CH₂-((2-CH₃)—Ph) 13 CH₂Ph CH₃ 14 ONC202 CH₂PhCH₂-((2-Cl)—Ph) 15 ONC203 CH₂Ph CH₂-(2-thienyl) 16 ONC204 CH₂Ph CH₂CH₂Ph17 ONC205 CH₂Ph CH₂CH₂(4-N- benzyl-piperazine) 18 ONC206 CH₂PhCH₂-(2,4-di F—Ph) 19 ONC207 H CH₂-((2-CH₃)—Ph) 20 ONC208 CH₃CH₂-((2-CH₃)—Ph) 21 ONC209 CH₂CH₂Ph CH₂-((2-CH₃)—Ph) 22 CH₂CH₂-(4-N-CH₂-((2-CH₃)—Ph) benzyl-piperizine) 23 CH₂CHOHPh CH₂-((2-CH₃)—Ph) 24(CH₂)₃CO—4F—Ph CH₂-((2-CH₃)—Ph) 32 ONC215 CH₂CH₂NHCOOC(CH₃)₃CH₂-((2-CH₃)—Ph) 33 ONC216 CH₂CH₂CH₂NH₂ CH₂-((2-CH₃)—Ph) 41 ONC210 CH₂PhCH₂-(3,5-di F—Ph) 51 ONC211 CH₂Ph CH₂-(3,4-di Cl—Ph) 52 ONC212 CH₂PhCH₂-(4-CF₃—Ph) 53 ONC213 CH₂Ph CH₂-(3,4-di F—Ph) 54 ONC214 CD₂C₆D₅CH₂-((2-CH₃)—Ph) 43 ONC217 CH₂Ph CH₂-(2-F—Ph) 55 ONC218 CH₂Ph CH₂(2-CH₃,4-F—Ph) 56 ONC219 CH₂Ph CH₂-(2,4-di Cl—Ph) 57 ONC220 CH₂PhCH₂-((4-OCH₃)—Ph) 34 ONC226 CH₂Ph CH₂-(3-pyridinyl) 35 ONC222 CH₂PhCH₂-(3-isoxazoliclinyl) 36 ONC224 CH₂Ph CH₂CH₂-(4-morpholinyl) 37 ONC223CH₂Ph CH₂-(4-CH₃—Ph) 38 ONC221 H CH₂-(4-CF₃—Ph) 73 ONC227 CH₂-(4-CF₃—Ph)CH₂-(4-CF₃—Ph) 72 ONC225 CH₂Ph CH₂-(2-F, 4-CF₃—Ph) 74 ONC228CH₂-(4-F—Ph) CH₂-(4-CF₃—Ph) 75 ONC229 CH₂-(OCH₃—Ph) CH₂-(4-CF₃—Ph) 76ONC230 (4-F—Ph)-4-oxobutyl CH₂-(4-CF₃—Ph) 77 ONC231 CH₂-3-pyridylCH₂-(4-CF₃—Ph) 78 ONC232 CH₂-4-methyl-2-thiazolyl CH₂-(4-CF₃—Ph) 79ONC233 CH₂-2-pyrazinyl CH₂-(4-CF₃—Ph) 81 ONC234 CH₂-(3,4-di Cl—Ph)CH₂-(4-CF₃—Ph) 82 ONC235 CH₂-(4-Cl—Ph) CH₂-(4-CF₃—Ph) 83 ONC236CH₂-3-thienyl CH₂-(4-CF₃—Ph) 84 ONC237 CH₂CH(OH)Ph CH₂-(4-CF₃—Ph)

IV. ASSESSING SENSITIVITY AND EFFICACY OF TREATMENT REGIMENS

Measuring expression, gene mutation, or gene copy number of a dopaminereceptor or another G protein-coupled receptor (GPCR) may be used topredict response or sensitivity to a method of treatment describedherein and to identify subjects likely to be responsive to a method oftreatment described herein, such as treatment with a compound of formula(10), a pharmaceutically acceptable salt thereof, or an analog thereof.In one aspect, provided herein are methods of identifying whether asubject having a condition is likely to be responsive to a treatmentregimen described herein. In one embodiment, the methods comprises (i)obtaining a biological sample from the subject; (ii) measuringexpression levels of at least one dopamine receptor or G protein-coupledreceptor (GPCR) in the sample; (iii) comparing the levels measured inthe sample to those for a pre-determined standard; and (iv) determiningwhether the subject is likely to be responsive to the treatment regimen,based on the levels measured in the sample to those for thepre-determined standard. In one embodiment, the step of measuring anexpression level of a dopamine receptor or GPCR in the sample includethe steps of (i) contacting the sample with an antibody orantigen-binding fragment that specifically binds to the receptor to forma complex of the antibody or antigen-binding fragment with the receptor;and (ii) measuring the amount of the complex. In one embodiment, thesubject has, or is at risk of having, cancer. In one embodiment, thecancer is a neuro-oncology disease. In one embodiment, the cancer is aneuroendocrine tumor. In one embodiment, the cancer is selected from thegroup consisting of meningioma, ependymoma, glioma, neuroblastoma, anddiffuse intrinsic pontine glioma. In one embodiment, the subject has, oris at risk of having, a psychiatric disorder. In one embodiment, thepsychiatric disorder is selected from the group consisting of psychosis,bipolar disorder, and major depressive disorder. In one embodiment, thesubject has, or is at risk of having, an infection. In one embodiment,the infection is a bacterial infection. In one embodiment, the infectionis a gram-negative bacterial infection. In one embodiment, the infectionis a gram-positive bacterial infection. In one embodiment, the bacterialinfection is an infection of a bacteria selected from the groupconsisting of Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, andEnterobacter species. In one embodiment, the gram-positive bacterialinfection is a Staphylococcus infection. In one embodiment, theStaphylococcus infection is an S. aureus infection (e.g., amethicillin-resistant S. aureus (MRSA) infection). In one embodiment,the treatment regimen comprises administering an effective amount of atherapeutic, such as a compound of formula (10), a pharmaceuticallyacceptable salt thereof, or an analog thereof. In one embodiment, thedopamine receptor is from the D2-like family of dopamine receptors Inone embodiment, the dopamine receptor is DRD2. In one embodiment, thedopamine receptor is DRD3. In one embodiment, the dopamine receptor isDRD4. In one embodiment, the dopamine receptor is DRD2, DRD3, or both.In one embodiment, the GPCR is a Class A GPCR. In one embodiment, theGPCR is GPR132. In one embodiment, the GPCR is selected from the groupconsisting of GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR,CXCR7, and combinations thereof. In one embodiment, the dopaminereceptor is DRD5, the treatment regimen comprises administering aneffective amount of a therapeutic, such as a compound of formula (10) ora pharmaceutically acceptable salt thereof, and an increased level ofexpression of DRD5 measured in the sample relative to the pre-determinedstandard indicates that the subject is or is not likely to be responsiveto the treatment regimen.

In another aspect, provided herein are methods of assessing theeffectiveness of a treatment regimen described herein, monitoring, orproviding a prognosis for a subject with a condition. In one embodiment,the methods comprises (i) obtaining a biological sample from thesubject; (ii) measuring expression levels of at least one dopaminereceptor or G protein-coupled receptor (GPCR) in the sample; (iii)comparing the levels measured in the sample to those for apre-determined standard; and (iv) determining a prognosis or determiningwhether the subject is responsive to the treatment regimen, based on thelevels measured in the sample to those for the pre-determined standard.In one embodiment, the step of measuring an expression level of adopamine receptor or GPCR in the sample include the steps of (i)contacting the sample with an antibody or antigen-binding fragment thatspecifically binds to the receptor to form a complex of the antibody orantigen-binding fragment with the receptor; and (ii) measuring theamount of the complex. In one embodiment, the methods comprise (i)obtaining a biological sample from the subject; (ii) measuring gene copynumber or mutations in at least one dopamine receptor in the sample;(iii) comparing the copy number measured or mutations found in thesample to those for a pre-determined standard; and (iv) determiningwhether the subject is responsive to the treatment regimen, based on thecopy number measured or mutations found in the sample to those for thepre-determined standard. In one embodiment, the subject has, or is atrisk of having, cancer. In one embodiment, the cancer is aneuro-oncology disease. In one embodiment, the cancer is aneuroendocrine tumor. In one embodiment, the cancer is selected from thegroup consisting of meningioma, ependymoma, glioma, neuroblastoma, anddiffuse intrinsic pontine glioma. In one embodiment, the subject has, oris at risk of having, a psychiatric disorder. In one embodiment, thepsychiatric disorder is selected from the group consisting of psychosis,bipolar disorder, and major depressive disorder. In one embodiment, thesubject has, or is at risk of having, an infection. In one embodiment,the infection is a bacterial infection. In one embodiment, the infectionis a gram-negative bacterial infection. In one embodiment, the infectionis a gram-positive bacterial infection. In one embodiment, the bacterialinfection is an infection of a bacteria selected from the groupconsisting of Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, andEnterobacter species. In one embodiment, the gram-positive bacterialinfection is a Staphylococcus infection. In one embodiment, theStaphylococcus infection is an S. aureus infection (e.g., amethicillin-resistant S. aureus (MRSA) infection). In one embodiment,the treatment regimen comprises administering an effective amount of atherapeutic, such as a compound of formula (10), a pharmaceuticallyacceptable salt thereof, or an analog thereof. In one embodiment, thedopamine receptor is selected from DRD2, DRD2S, DRD2L, and DRD3. In oneembodiment, the dopamine receptor is from the D2-like family of dopaminereceptors. In one embodiment, the dopamine receptor is from the D1-likefamily of dopamine receptors. In one embodiment, the dopamine receptoris DRD1. In one embodiment, the dopamine receptor is DRD2. In oneembodiment, the dopamine receptor is DRD3. In one embodiment, thedopamine receptor is DRD4. In one embodiment, the dopamine receptor isDRD5. In one embodiment, the dopamine receptor is DRD2, DRD3, or both.In one embodiment, the GPCR is a Class A GPCR. In one embodiment, theGPCR is GPR132. In one embodiment, the GPCR is selected from the groupconsisting of GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR,CXCR7, and combinations thereof.

In one embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a compound of formula(10) or a pharmaceutically acceptable salt thereof, and an increasedlevel of expression of DRD5 measured in the sample relative to thepre-determined standard indicates that the treatment regimen is or isnot effective. In one embodiment, the dopamine receptor is DRD5, thetreatment regimen comprises administering an effective amount of atherapeutic, such as a compound of formula (10) or a pharmaceuticallyacceptable salt thereof, and mutation in the DRD5 gene measured in thesample indicates that the treatment regimen is or is not effective. Inone embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10) or a pharmaceutically acceptable salt thereof,and the misense mutation Q366R in the DRD5 gene measured in the sampleindicates that the treatment regimen is or is not effective.

In another aspect, provided herein are methods of identifying whether asubject having a condition is likely to be responsive to a treatmentregimen described herein. In one embodiment, the methods comprises (i)obtaining a biological sample from the subject; (ii) measuring gene copynumber or mutations in at least one dopamine receptor in the sample;(iii) comparing the copy number measured or mutations found in thesample to those for a pre-determined standard; and (iv) determiningwhether the subject is likely to be responsive to the treatment regimen,based on the copy number measured or mutations found in the sample tothose for the pre-determined standard. In one embodiment, the subjecthas, or is at risk of having, cancer. In one embodiment, the cancer is aneuro-oncology disease. In one embodiment, the cancer is aneuroendocrine tumor. In one embodiment, the cancer is selected from thegroup consisting of meningioma, ependymoma, glioma, neuroblastoma, anddiffuse intrinsic pontine glioma. In one embodiment, the subject has, oris at risk of having, a psychiatric disorder. In one embodiment, thepsychiatric disorder is selected from the group consisting of psychosis,schizophrenia, bipolar disorder, and major depressive disorder. In oneembodiment, the subject has, or is at risk of having, an infection. Inone embodiment, the infection is a bacterial infection. In oneembodiment, the infection is a gram-negative bacterial infection. In oneembodiment, the infection is a gram-positive bacterial infection. In oneembodiment, the bacterial infection is an infection of a bacteriaselected from the group consisting of Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,Pseudomonas aeruginosa, and Enterobacter species. In one embodiment, thegram-positive bacterial infection is a Staphylococcus infection. In oneembodiment, the Staphylococcus infection is an S. aureus infection(e.g., a methicillin-resistant S. aureus (MRSA) infection). In oneembodiment, the treatment regimen comprises administering an effectiveamount of a therapeutic, such as a compound of formula (10), apharmaceutically acceptable salt thereof, or an analog thereof. In oneembodiment, the dopamine receptor is from the D2-like family of dopaminereceptors. In one embodiment, the dopamine receptor is DRD1. In oneembodiment, the dopamine receptor is DRD2. In one embodiment, thedopamine receptor is DRD3. In one embodiment, the dopamine receptor isDRD4. In one embodiment, the dopamine receptor is DRD5. In oneembodiment, the dopamine receptor is DRD2, DRD3, or both. In oneembodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10) or a pharmaceutically acceptable salt thereof,and mutation in the DRD5 gene measured in the sample indicates that thesubject is or is not likely to be responsive to the treatment regimen.In one embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10) or a pharmaceutically acceptable salt thereof,and the misense mutation Q366R in the DRD5 gene measured in the sampleindicates that the subject is or is not likely to be responsive to thetreatment regimen.

In addition, measuring expression, post-translational modifications, oractivity levels of or mutations in eIF2-α, ATF4, CHOP, DR5, or cleavedor total cytokeratin 18 may be used to predict response or sensitivityto a method of treatment described herein and to identify subjectslikely to be responsive to a method of treatment described herein, suchas treatment with a compound of formula (10), a pharmaceuticallyacceptable salt thereof, or an analog thereof. In addition, measuringexpression, post-translational modifications, or activity levels of ormutations in eIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18can be used to assess the effectiveness of or monitor a method oftreatment described herein. Furthermore, measuring expression,post-translational modifications, or activity levels of or mutations ineIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be usedto screen in vivo, in vitro, or in silico for structurally unrelatedanti-cancer molecules. For example, competition and other assays knownin the art may be used to identify drugs able to outcompete the targetinteraction with a higher affinity to compare changes in those levels tothe respective changes produced by a compound of formula (10) or ananalog thereof. Assays can also be performed on living mammalian cells,which more closely approximate the effects of a particular serum levelof drug in the body, or on microsomal extracts prepared from culturedcell lines.

In one embodiment, the subject has, or is at risk of having, cancer. Inone embodiment, the treatment regimen comprises administering aneffective amount of an imipridone, such as ONC201, or an analog thereof.In one embodiment, the treatment regimen comprises administering aneffective amount of ONC201. In one embodiment, the treatment regimencomprises administering an effective amount of a compound of formula(10). In one embodiment, the compound of formula (10) is a compound offormula (40), e.g., a compound of formula (45). In one embodiment, acompound of formula (10) is a compound of formula (50), e.g., a compoundformula (55). In one embodiment, the compound of formula (10) is acompound of formula (80). In one embodiment, the compound of formula(10) is a compound of formula (90). In one embodiment, the compound offormula (10) is a compound of formula (60). In one embodiment, analogsof compound (1) have a structure selected from the structures of formula(25), formula (26), formula (27), formula (28), formula (29), formula(30), or formula (31).

Levels for a pre-determined standard can be, e.g., the average or medianlevels measured in samples from subjects. The levels for apre-determined standard can be measured under the same or substantiallysimilar experimental conditions as in measuring a sample from a subject.The levels for the pre-determined standard may be obtained from subjectswho are responsive to treatment with an imipridone, such as ONC201, oran analog thereof. In one embodiment, the pre-determined standard isobtained from subjects who are responsive to treatment with thecompound, and if the levels in a sample from a subject are similar tothose in the standard, then the subject can be classified as likely tobe responsive to treatment. The levels for the pre-determined standardmay be obtained from subjects who are not responsive to treatment withthe compound. In one embodiment, the pre-determined standard is obtainedfrom subjects who are not responsive to treatment with the compound, andif the levels in a sample from a subject are different (e.g., up- ordown-regulated) than in the pre-determined standard, then the subjectcan be classified as likely to be responsive to treatment. The levelsfor the pre-determined standard may be obtained from normal healthysubjects.

Immunoassays can be used to assay protein or methylation levels in asample, including enzyme-linked immunosorbent assay (ELISA),enzyme-linked immunofiltration assay (ELIFA), flow cytometry,immunoblot, immunoprecipitation, immunohistochemistry,immunocytochemistry, luminescent immunoassay (LIA), fluorescentimmunoassay (FIA), and radioimmunoassay. m⁶A mRNA methylation levels canbe obtained by methylated RNA immunoprecipitation (Me-RIP)) or otherquantitative biochemical assays known in the art.

Nucleic acid mutations can be determined by any of a number of knownprocedures. For example, a biologic sample from an individual can firstbe obtained. Such biological samples include, but are not limited to, abodily fluid (such as urine, saliva, plasma, or serum) or a tissuesample (such as a buccal tissue sample or buccal cell). The biologicsample can then be sequenced or scanned using known methods. Forexample, DNA arrays can be used to analyze at least a portion of thesubject's genomic sequence. Furthermore, whole or partial genomesequence information can be used. Such sequences can be determined usingstandard sequencing methods including chain-termination (Sangerdideoxynucleotide), dye-terminator sequencing, and SOLID™ sequencing(Applied Biosystems). Whole genome sequences can be cut by restrictionenzymes or sheared (mechanically) into shorter fragments for sequencing.DNA sequences can also be amplified using known methods such as PCR andvector-based cloning methods (e.g., Escherichia coli). In oneembodiment, at least a portion of a subject's genetic material (e.g.,DNA, RNA, mRNA, cDNA, other nucleotide bases or derivatives of these) isscanned or sequenced using, e.g., conventional DNA sequencers orchip-based technologies, to identify the presence or absence ofmutations or copy number variations.

In one aspect, provided herein are methods of identifying and treating asubject having a condition and who is likely to be responsive to atreatment regimen described herein. In one embodiment, the methodcomprises (i) identifying whether a subject having a condition is likelyto be responsive to a treatment regimen described herein; and (ii)treating with the treatment regimen a subject determined likely to beresponsive to that treatment regimen. In one embodiment, the subjecthas, or is at risk of having, cancer. In one embodiment, the treatmentregimen comprises administering an effective amount an imipridone, e.g.,ONC201 or an analog thereof. In one embodiment, the treatment regimencomprises administering an effective amount of compound (1). In oneembodiment, the treatment regimen comprises administering an effectiveamount of a compound of formula (10). In one embodiment, the compound offormula (10) is a compound of formula (40), e.g., a compound of formula(45). In one embodiment, a compound of formula (10) is a compound offormula (50), e.g., a compound formula (55). In one embodiment, thecompound of formula (10) is a compound of formula (80). In oneembodiment, the compound of formula (10) is a compound of formula (90).In one embodiment, the compound of formula (10) is a compound of formula(60). In one embodiment, analogs of compound (1) have a structureselected from the structures of formula (25), formula (26), formula(27), formula (28), formula (29), formula (30), or formula (31).

Levels for a pre-determined standard can be, e.g., the average or medianlevels measured in samples from subjects. The levels for apre-determined standard can be measured under the same or substantiallysimilar experimental conditions as in measuring a sample from a subject.The levels for the pre-determined standard may be obtained from subjectswho are responsive to treatment with an imipridone, such as ONC201 or ananalog thereof. In one embodiment, the pre-determined standard isobtained from subjects who are responsive to treatment with thecompound, and if the levels in a sample from a subject are similar tothose in the standard, then the subject can be classified as likely tobe responsive to treatment. The levels for the pre-determined standardmay be obtained from subjects who are not responsive to treatment withthe compound. In one embodiment, the pre-determined standard is obtainedfrom subjects who are not responsive to treatment with the compound, andif the levels in a sample from a subject are different (e.g., up- ordown-regulated) than those in the pre-determined standard, then thesubject can be classified as likely to be responsive to treatment. Thelevels for the pre-determined standard may be obtained from normalhealthy subjects. Immunoassays can be used to assay protein levels in asample.

In one aspect, provided herein are methods of treating and assessing theeffectiveness of a treatment in a subject having a condition. In oneembodiment, the method comprises (i) treating the subject according to amethod of treatment described herein (ii) assessing as described hereinthe effectiveness of the treatment. In one embodiment, the subject has,or is at risk of having, cancer. In one embodiment, the treatmentregimen comprises administering an effective amount of an imipridone,such as ONC201 or an analog thereof. In one embodiment, the treatmentregimen comprises administering an effective amount of compound (1). Inone embodiment, the treatment regimen comprises administering aneffective amount of a compound of formula (10). In one embodiment, thecompound of formula (10) is a compound of formula (40), e.g., a compoundof formula (45). In one embodiment, a compound of formula (10) is acompound of formula (50), e.g., a compound formula (55). In oneembodiment, the compound of formula (10) is a compound of formula (80).In one embodiment, the compound of formula (10) is a compound of formula(90). In one embodiment, the compound of formula (10) is a compound offormula (60). In one embodiment, analogs of compound (1) have astructure selected from the structures of formula (25), formula (26),formula (27), formula (28), formula (29), formula (30), or formula (31).

Other conditions that may be suitable for the methods described hereininclude Attention Deficit Disorder; Addiction; Epilepsy; Viralinfection; Inflammation; Neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, Amyotrophic lateralsclerosis; Cardiovascular diseases such as coronary artery disease,cardiomyopathy, hypertensive heart disease, heart failure, pulmonaryheart disease, cardiac dysrhythmias, inflammatory heart disease,endocarditis, inflammatory cardiomegaly, myocarditis, valvular heartdisease, cerebrovascular disease, peripheral arterial disease,congenital heart disease, rheumatic heart disease; Diabetes; and lightchain amyloidosis.

V. COMPOSITIONS

In one aspect, pharmaceutical compositions are provided, comprisingcompounds of formula (10):

or of formula (1):

and their pharmaceutically acceptable salts In one embodiment, the saltis a pharmaceutically acceptable mono-salt of the compound. In oneembodiment, the salt is a pharmaceutically acceptable di-salt of thecompound. In one embodiment, the salt is a pharmaceutically acceptablemono- or multi-salt (e.g., a di-salt or tri-salt) thereof selected fromthe group consisting of hydrochloride, hydrobromide, hydrogensulphate,sulfates, phosphates, fumarates, succinates, oxalates and lactates,bisulfates, hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate,malate, maleate, fumarate sulfonate, methylsulfonate, formate, acetate,and carboxylate. In one embodiment, the salt is a salt selected from thegroup consisting of p-toluene-sulfonate, benzenesulfonate, citrate,methanesulfonate, oxalate, succinate, tartrate, fumarate and maleate. Inone embodiment, the salt is a salt selected from the group consisting ofammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/orwith counter-ions such as methylamino, dimethylamino, diethylamino andtriethylamino counter-ions. In one embodiment, the salt is a.di-hydrochloride salt or a di-hydrobromide salt.

Compound (1) (ONC201) has the same chemical structure that would berevealed by structural analysis (e.g., NMR, X-ray diffraction) ofcompound NSC 350625, available from the National Cancer Institute'sDevelopmental Therapeutics Program Repository.

In one embodiment, the pharmaceutical composition includes a di-salt(e.g., a di-hydrochloride salt) of ONC201 or an analog thereof (e.g., animipridone). Salts (e.g., di-salts or tri-salts) of an ONC201 analog canbe prepared from an ONC201 analog, which can be synthesized as describedherein, or using standard chemical synthetic methodology known to one ofordinary skill in the art.

In one embodiment, the pharmaceutical composition includes at least onepharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers, include, but are not limited to, those in Handbookof Pharmaceutical Excipients, 7^(th) ed., edited by Raymond C. Rowe etal., American Pharmaceutical Association, Washington, USA andPharmaceutical Press, London; and earlier editions. Exemplarypharmaceutically acceptable carriers, methods for making pharmaceuticalcompositions and various dosage forms, as well as administration modesare well-known in the art, for example as detailed in PharmaceuticalDosage Forms: Tablets, edited by Larry L. Augsburger & Stephen W. Hoag.,London: Informa Healthcare, 2008; and in L. V. Allen, Jr. et al.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8^(th)ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004; A. R.Gennaro, Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins, 21^(st) ed., 2005, particularly chapter 89; and J.G. Hardman et al., Goodman & Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill Professional, 10^(th) ed., 2001.

In one embodiment, pharmaceutical compositions are formulated for ocularadministration. In one embodiment, pharmaceutical compositions areformulated for topical administration. In one embodiment, pharmaceuticalcompositions are formulated as drops, ointments, or liquids. In oneembodiment, pharmaceutical compositions include conventionalpharmaceutical carriers such as aqueous, powdery or oily bases,thickeners.

In one embodiment, a pharmaceutical composition is a formulation forintravenous administration. In one embodiment, the intravenousformulation comprises a compound of formula (10) or a pharmaceuticallyacceptable salt thereof dissolved in a solvent. In one embodiment, thesolvent comprises water. In one embodiment, the intravenous formulationincludes the compound or its salt in a concentration of about 0.05,about 0.25, about 0.5, about 2.5, about 5, about 25, or about 50 mg/mL.In one embodiment, the intravenous formulation includes the compound orits salt in a concentration of from about 0.05, 0.5, or 5 mg/mL to about1, 10, or 100 mg/mL. In one embodiment, the intravenous formulationincludes from about 0.005% 0.05%, or 0.5% to about 0.1%, 1%, or 10% ofthe compound or its salt. In one embodiment, the intravenous formulationincludes about 0.05%, 0.5%, or 5% of the compound or its salt. In oneembodiment, the intravenous formulation includes a higher or a lowerconcentration of the compound or its salt.

In one embodiment, the intravenous formulation has a pH of about 3. Inone embodiment, the formulation is adjusted to pH 3 with a phosphatebuffer. In one embodiment, the intravenous formulation includes dextroseor sodium chloride. In one embodiment, the intravenous formulationincludes the compound or its salt in a concentration of about 5 mg/mLand pH 3 and forms a stable solution. In one embodiment, the intravenousformulation includes the compound or its salt in a concentration ofabout 5 mg/mL and pH<5 and forms a stable solution. In one embodiment,the intravenous formulation includes the compound or its salt and one ormore antioxidants. In one embodiment, the intravenous formulationincludes a mixture of mono- and di-hydrochloride salts of the compound.In one embodiment, the intravenous formulation includes the compound orits salt as a 1% solution in a concentration of about 10 mg/mL. Forexample, the intravenous formulation is a solution with a pH of about3.3. In one embodiment, the pH is less than 4.0.

In one embodiment, the pharmaceutical composition further includes apharmaceutically acceptable carrier. In one embodiment, a suitablepharmaceutically acceptable carrier includes an aqueous carrier. In oneembodiment, the aqueous carrier includes sterile water. In oneembodiment, the formulation includes dextrose and/or sodium. In oneembodiment, the pharmaceutically acceptable carrier includes an oil.

In one embodiment, an intravenous formulation comprises ONC201 or ananalog thereof or a di-hydrochloride salt thereof dissolved in water at25 mg/mL. In one embodiment, the formulation is adjusted to pH 3 withphosphate buffer. In one embodiment, the formulation includes dextrose,sodium chloride or both. In one embodiment, the formulation includes ahigher or a lower concentration of the di-hydrochloride salt of ONC201or an analog thereof. In one embodiment, the formulation includes ONC201or an analog thereof or a di-hydrochloride salt thereof in aconcentration of about 5 mg/mL. In one embodiment, the formulation ofabout 5 mg/mL forms a stable solution and pH 3. In one embodiment, theformulation of about 5 mg/mL has a pH<5 and forms a stable solution. Inone embodiment, the intravenous formulation includes ONC201 or an analogthereof or a di-hydrochloride salt thereof and one or more antioxidants.In one embodiment, the intravenous formulation includes a mixture ofmono- and di-hydrochloride salts of ONC201 or an analog thereof. In oneembodiment, the intravenous formulation includes ONC201 or an analogthereof or a di-hydrochloride salt thereof as a 1% solution in aconcentration of about 10 mg/mL. For example, the intravenousformulation is a solution having a pH of about 3.3. In one embodiment,the pH is less than 4.0.

In one embodiment, the intravenous formulation includes from about 0.5%to about 10% (or from about 5 mg/mL to about 100 mg/mL) of ONC201 or ananalog thereof or a di-salt thereof. In one embodiment, the formulationincludes from about 5% (or about 50 mg/mL) of ONC201 or an analogthereof or a di-salt thereof. In one embodiment, the intravenousinfusion rate may be slowed to decrease side effects of ONC201 or ananalog thereof or a di-salt thereof.

In one embodiment, the pharmaceutical composition comprises about0.1-99% of an ONC201 salt or an analog thereof; and a pharmaceuticallyacceptable carrier, e.g., an oil or sterile water or other aqueouscarrier. In one embodiment, the composition comprises a mono or di-saltof ONC201 or an analog thereof in a range of from about 5% to about 50%for oral dosage forms.

In one embodiment, a pharmaceutical composition includes an antioxidant.Suitable antioxidants include: ascorbic acid derivatives such asascorbic acid, erythorbic acid, sodium ascorbate, thiol derivatives suchas thioglycerol, cysteine, acetylcysteine, cystine, dithioerythritol,dithiothreitol, glutathione, tocopherols, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), sulfurous acid salts such assodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodiummetabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, andsodium thiosulfate, nordihydroguaiaretic acid. It should be noted thatantioxidants used for aqueous formulations typically include: sodiumsulphite, sodium metabisulphite, sodium formaldehyde sulphoxylate andascorbic acid and combinations thereof, whereas antioxidants used inoil-based solutions, organic solvents, include butylated hydroxytoluene(BHT), butylated hydroxyanisole (BHA) and propyl gallate andcombinations thereof. In yet other embodiments, an antioxidant can beone or more of a flavanoid, an isoflavone, monothioglycerol, L-cysteine,thioglycolic acid, α-tocopherol, ascorbic acid 6-palmitate,dihydrolipoic acid, butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), vitamin E, propyl gallate, β-carotene, ascorbicacid. Antioxidants can typically be used in about 0.1% to 1.0% byweight, more typically about 0.2%.

In one embodiment, the pharmaceutical composition includes animipridone, such as ONC201 or an analog thereof, or a pharmaceuticallyacceptable salt thereof and at least one other therapeutic agent. Forexample, the other therapeutic agent is selected from the groupconsisting of hormone analogs and antihormones, aromatase inhibitors,LHRH agonists and antagonists, inhibitors of growth factors, growthfactor antibodies, growth factor receptor antibodies, tyrosine kinaseinhibitors; antimetabolites; antitumour antibiotics; platinumderivatives; alkylation agents; antimitotic agents; tubuline inhibitors;PARP inhibitors, topoisomerase inhibitors, serine/threonine kinaseinhibitors, tyrosine kinase inhibitors, protein protein interactioninhibitors, RAF inhibitors, MEK inhibitors, ERK inhibitors, IGF-1Rinhibitors, ErbB receptor inhibitors, rapamycin analogs, BTK inhibitors,CRM1 inhibitors (e.g., KPT185), P53 modulators (e.g., Nutlins),antiangiogenics (e.g., axitinib, aflibercept, sorafenib, andregorafenib), amifostin, anagrelid, clodronat, filgrastin, interferon,interferon α, leucovorin, rituximab, procarbazine, levamisole, mesna,mitotane, pamidronate and porfimer, 2-chlorodesoxyadenosine,2-fluorodesoxy-cytidine, 2-methoxyoestradiol, 2C4,3-alethine,131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B,A 105972, A 204197, abiraterone, aldesleukin, alitretinoin,allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole,AG-2037, AP-5280, apaziquone, apomine, aranose, arglabin, arzoxifene,atamestane, atrasentan, auristatin PE, ABT-199 (Venetoclax), ABT-263(Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162,ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (selumetinib),ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055,AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib), avastin,AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate,BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797,BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992(afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI 6727,BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid, bleomycin A,bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin,busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell, calcitriol, canertinib,canfosfamide, capecitabine, carboxyphthalatoplatin, CC1-779, CC-115,CC-223, CEP-701, CEP-751, CBT-1 cefixime, ceflatonin, ceftriaxone,celecoxib, celmoleukin, cemadotin, CH4987655/RO-4987655,chlorotrianisene, cilengitide, ciclosporin, CDA-II, CDC-394, CKD-602,CKI-27, clofarabin, colchicin, combretastatin A4, COT inhibitors,CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin 52, CTP-37, CTLA-4monoclonal antibodies, CP-461, CV-247, cyanomorpholinodoxorubicin,cytarabine, D 24851, decitabine, deoxorubicin, deoxyrubicin,deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone,dexrazoxanet, diethylstilbestrol, diflomotecan, didox, DMDC, dolastatin10, doranidazole, DS-7423, E7010, E-6201, edatrexat, edotreotide,efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509,enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab,ER-86526, erlotinib, ET-18-0CH3, ethynylcytidine, ethynyloestradiol,exatecan, exatecan mesylate, exemestane, exisulind, fenretinide,figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI,formestane, fotemustine, galarubicin, gallium maltolate, gefinitib,gemtuzumab, gimatecan, glufosfamide, GCS-100, GDC-0623, GDC-0941(pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DTimmunogen, GMK, GPX-100, gp100-peptide vaccines, GSK-5126766,GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib),GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795,GW2016, granisetron, herceptine, hexamethylmelamine, histamine,homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesteronecaproate, ibandronate, ibrutinib, ibritumomab, idatrexate, idenestrol,IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), immunol,indisulam, interferon α-2a, interferon α-2b, pegylated interferon α-2b,interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab,iproplatin, irofulven, isohomohalichondrin-B, isoflavone, isotretinoin,ixabepilone, JRX-2, JSF-154, J-107088, conjugated oestrogens, kahalid F,ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim,leuprolide, leuporelin, lexidronam, LGD-1550, linezolid, lutetiumtexaphyrin, lometrexol, losoxantrone, LU 223651, lurtotecan, LY-S6AKT1,LY-2780301, mafosfamide, marimastat, mechloroethamine, MEK inhibitors,MEK-162, methyltestosteron, methylprednisolone, MEDI-573, MEN-10755,MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid,mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, O6-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,regorafenib, revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440,rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab,rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone,rubitecan, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA,sargramostim, satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668,SDX-101, semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897,SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment, the other therapeutic agent comprises a hormoneanalog, an antihormone or both selected from tamoxifen, toremifene,raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide,bicalutamide, aminoglutethimide, cyproterone acetate, finasteride,buserelin acetate, fludrocortisone, fluoxymesterone,medroxy-progesterone, octreotide, and combinations thereof. In oneembodiment, the other therapeutic agent comprises one or more LHRHagonists and/or antagonists selected from goserelin acetate, luprolideacetate, triptorelin pamoate and combinations thereof and wherein theLHRH antagonists are selected from Degarelix, Cetrorelix, Abarelix,Ozarelix, Degarelix combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more growth factor inhibitorsselected from inhibitors of: platelet derived growth factor (PDGF),fibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), insuline-like growth factors(IGF), human epidermal growth factor (HER) and hepatocyte growth factor(HGF). In one embodiment, the other therapeutic agent comprises one ormore inhibitors of the human epidermal growth factor selected from HER2,HER3, and HER4. In one embodiment, the other therapeutic agent comprisesone or more tyrosine kinase inhibitors selected from cetuximab,gefitinib, imatinib, lapatinib and trastuzumab, and combinationsthereof. In one embodiment, the other therapeutic agent comprises one ormore aromatase inhibitors selected from anastrozole, letrozole,liarozole, vorozole, exemestane, atamestane, and combinations thereof.In one embodiment, the other therapeutic agent comprises one or moreantimetabolites which are antifolates selected from methotrexate,raltitrexed, and pyrimidine analogs. In one embodiment, the othertherapeutic agent comprises one or more antimetabolites which arepyrimidine analogs selected from 5-fluorouracil, capecitabin andgemcitabin. In one embodiment, the other therapeutic agent comprises oneor more antimetabolites which are purine and/or adenosine analogsselected from mercaptopurine, thioguanine, cladribine and pentostatin,cytarabine, fludarabine, and combinations thereof. In one embodiment,the other therapeutic agent comprises one or more antitumour antibioticsselected from anthracyclins, doxorubicin, daunorubicin, epirubicin andidarubicin, mitomycin-C, bleomycin, dactinomycin, plicamycin,streptozocin and combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more platinum derivatives selectedfrom cisplatin, oxaliplatin, carboplatin and combinations thereof. Inone embodiment, the other therapeutic agent comprises one or morealkylation agents selected from estramustin, meclorethamine, melphalan,chlorambucil, busulphan, dacarbazin, cyclophosphamide, ifosfamide,temozolomide, nitrosoureas, and combinations thereof. In one embodiment,the other therapeutic agent comprises nitrosoureas selected fromcarmustin, lomustin, thiotepa, and combinations thereof. In oneembodiment, the other therapeutic agent comprises antimitotic agentsselected from Vinca alkaloids and taxanes. In one embodiment, the othertherapeutic agent comprises one or more taxanes selected frompaclitaxel, docetaxel, and combinations thereof. In one embodiment, theother therapeutic agent comprises one or more Vinca alkaloids selectedfrom vinblastine, vindesin, vinorelbin, vincristine, and combinationsthereof. In one embodiment, the other therapeutic agent comprises one ormore topoisomerase inhibitors which are epipodophyllotoxins. In oneembodiment, the other therapeutic agent comprises one or moreepipodophyllotoxins selected from etoposide and etopophos, teniposide,amsacrin, topotecan, irinotecan, mitoxantron, and combinations thereof.In one embodiment, the other therapeutic agent comprises one or moreserine/threonine kinase inhibitors selected from PDK 1 inhibitors, B-Rafinhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dualmTOR/PI3K inhibitors, STK 33 inhibitors, AKT inhibitors, PLK 1inhibitors, inhibitors of CDKs, Aurora kinase inhibitors, andcombinations thereof. In one embodiment, the other therapeutic agentcomprises one or more tyrosine kinase inhibitors which are PTK2/FAKinhibitors. In one embodiment, the other therapeutic agent comprises oneor more protein protein interaction inhibitors selected from IAP, Mcl-1,MDM2/MDMX and combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more rapamycin analogs selected fromeverolimus, temsirolimus, ridaforolimus, sirolimus, and combinationsthereof. In one embodiment, the other therapeutic agent comprises one ormore therapeutic agents selected from amifostin, anagrelid, clodronat,filgrastin, interferon, interferon α, leucovorin, rituximab,procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, andcombinations thereof. In one embodiment, the other therapeutic agentcomprises one or more therapeutic agents selected from2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyoestradiol,2C4,3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin,16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin,alitretinoin, allovectin-7, altretamine, alvocidib, amonafide,anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, aranose,arglabin, arzoxifene, atamestane, atrasentan, auristatin PE, ABT-199(Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479(ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244(selumetinib), ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717,AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib),avastin, AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006,BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodardicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550,BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid,bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib,brostallicin, busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell,calcitriol, canertinib, canfosfamide, capecitabine,carboxyphthalatoplatin, CC1-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1cefixime, ceflatonin, ceftriaxone, celecoxib, celmoleukin, cemadotin,CH4987655/RO-4987655, chlorotrianisene, cilengitide, ciclosporin,CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicin, combretastatinA4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin52, CTP-37, CTLA-4 monoclonal antibodies, CP-461, CV-247,cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine,deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide,desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol,diflomotecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010,E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFRinhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin,epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3,ethynylcytidine, ethynyloestradiol, exatecan, exatecan mesylate,exemestane, exisulind, fenretinide, figitumumab, floxuridine, folicacid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin,gallium maltolate, gefinitib, gemtuzumab, gimatecan, glufosfamide,GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068,GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100-peptidevaccines, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436(dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183,GSK-2141795, GW2016, granisetron, herceptine, hexamethylmelamine,histamine, homoharringtonine, hyaluronic acid, hydroxyurea,hydroxyprogesterone caproate, ibandronate, ibrutinib, ibritumomab,idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12(cixutumumab), immunol, indisulam, interferon α-2a, interferon α-2b,pegylated interferon α-2b, interleukin-2, INK-1117, INK-128, INSM-18,ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B,isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088,conjugated oestrogens, kahalid F, ketoconazole, KW-2170, KW-2450,lobaplatin, leflunomide, lenograstim, leuprolide, leuporelin,lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol,losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide,marimastat, mechloroethamine, MEK inhibitors, MEK-162,methyltestosteron, methylprednisolone, MEDI-573, MEN-10755, MDX-H210,MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin,mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb,rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453,RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan,R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim,satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101,semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747,SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment, the other therapeutic agent comprises a steroid,including dexamethasone, prednisolone, methyl prednisolone, prednisone,hydrocortisone, triamcinolone, betamethasone, and cortivazol. In oneembodiment, the other therapeutic agent comprises an anti-emetic,Anti-emetics include, but are not limited to, 5-HT3 receptor agonists(e.g., dolasetron, granisetron, ondansetron, tropisetron, palonosetron,and mirtazapine), dopamine agonists (e.g., domperidone, olanzapine,droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride,prochlorperazine, and metoclopramide), NK1 receptor antagonists (e.g.,aprepitant and casopitant), antihistamines (such as cyclizine,diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine,hydroxyzine), cannabinoids (e.g., cannabis, dronabinol, nabilone, andsativex), benzodiazepines (e.g., midazolam and lorazepam),anticholinergics (e.g., hyoscine), trimethobenzamide, ginger, emetrol,propofol, peppermint, muscimol, and ajwain.

In one embodiment, the other therapeutic agent comprises an anti-canceragent, which includes a mitotic inhibitor. In one embodiment, themitotic inhibitor includes a taxane. In one embodiment, the mitoticinhibitor includes a taxane selected from paclitaxel and docetaxel.

In one embodiment, the pharmaceutical composition includes animipridone, such as ONC201, or an analog thereof, or a pharmaceuticallyacceptable salt thereof; and at least one anti-cancer agent, whichincludes one or more of acivicin, aclarubicin, acodazole, acronine,adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine,ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine,anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin,azacitidine, azetepa, azotomycin, batimastat, benzodepa, bevacizumab,bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin,brequinar, bropirimine, busulfan, cactinomycin, calusterone,capecitabine, caracemide, carbetimer, carboplatin, carmustine,carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin,dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin,droloxifene, dromostanolone, duazomycin, edatrexate, elformithine,elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin,erbulozole, esorubicin, estramustine, etanidazole, etoposide, etoprine,fadrozole, fazarabine, fenretinide, floxuridine, fludarabine,fluorouracil, flurocitabine, fosquidone, fostriecin, fulvestrant,gemcitabine, hydroxyurea, idarubicin, ifosfamide, ilmofosine,interleukin II (IL-2, including recombinant interleukin II or rIL2),interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3,interferon β-Ia, interferon gamma-Ib, iproplatin, irinotecan,lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine,losoxantrone, masoprocol, maytansine, mechlorethamine hydrochloride,megestrol, melengestrol acetate, melphalan, menogaril, mercaptopurine,methotrexate, metoprine, meturedepa, mitindomide, mitocarcin,mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane,mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogalamycin,ormnaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin,pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan,piroxantrone hydrochloride, plicamycin, plomestane, porfimer,porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin,riboprine, rogletimide, safingol, semustine, simtrazene, sparfosate,sparsomycin, spirogermanium, spiromustine, spiroplatin, streptonigrin,streptozocin, sulofenur, talisomycin, tamoxifen, tecogalan, tegafur,teloxantrone, temoporfin, teniposide, teroxirone, testolactone,thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan,toremifene, trestolone, triciribine, trimetrexate, triptorelin,tubulozole, uracil mustard, uredepa, vapreotide, verteporfin,vinblastine, vincristine sulfate, vindesine, vinepidine, vinglycinate,vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole,zeniplatin, zinostatin, zoledronate, zorubicin and combinations thereof.

Examples of suitable anti-cancer agents include those described Goodmanand Gilman's The Pharmacological Basis of Therapeutics, 12^(th) Ed.,edited by Laurence Brunton, Bruce Chabner, Bjorn Knollman, McGraw HillProfessional, 2010.

In some exemplary embodiments, the pharmaceutical composition includes asalt (e.g., a mono- or di-salt) of an imipridone, e.g., ONC201, or ananalog thereof and at least one other therapeutic agent, where the othertherapeutic agent comprises an anti-angiogenic agent, for example,bevacizumab. In one embodiment, the anti-angiogenic agent is selectedfrom aflibercept, axitinib, angiostatin, endostatin, 16 kDa prolactinfragment, laminin peptides, fibronectin peptides, tissuemetalloproteinase inhibitors (TIMP 1, 2, 3, 4), plasminogen activatorinhibitors (PAI-1, -2), tumor necrosis factor α, (high dose, invitro),TGF-β1, interferons (IFN-α, -β, γ), ELR-CXC chemokines, IL-12; SDF-1;MIG; platelet factor 4 (PF-4); IP-10, thrombospondin (TSP), SPARC,2-methoxyoestradiol, proliferin-related protein, suramin, sorafenib,regorafenib, thalidomide, cortisone, linomide, fumagillin (AGM-1470;TNP-470), tamoxifen, retinoids, CM101, dexamethasone, leukemiainhibitory factor (LIF), hedgehog inhibitor and combinations thereof.

A pharmaceutical combination can include first and second therapeuticagents in any desired proportions provided that the synergistic orcooperative effect still occurs. A synergistic pharmaceuticalcombination preferably contains the first and second therapeutic agentsin a ratio of from about 1:9 to about 9:1. In one embodiment, asynergistic combination contains the first and second therapeutic agentsin a ratio of from about 1:8 to about 8:1, from about 1:7 to about 7:1,from about 1:6 to about 6:1, from about 1:5 to about 5:1, from about 1:4to about 4:1, from about 1:3 to about 3:1, or from about 1:2 to about2:1. In one embodiment, the synergistic combination contains thetherapeutic agents in a ratio of approximately 1:1.

In one embodiment, the second therapeutic agent is selected fromAllopurinol, Arsenic Trioxide, Azacitidine, Bortezomib, Bevacizumab,Capecitabine, Carboplatin, Celecoxib, Chlorambucil, Clofarabine,Cytarabine, Dacarbazine, Daunorubicin HCl, Docetaxel, Doxorubicin HCl,Floxuridine, Gemcitabine HCl, Hydroxyurea, Ifosfamide, ImatinibMesylate, Ixabepilone, Lenalidomide, Megestrol acetate, Methotrexate,Mitotane, Mitoxantrone HCl, Oxaliplatin, Paclitaxel, Pralatrexate,Romidepsin, Sorafenib, Streptozocin, Tamoxifen Citrate, Topotecan HCl,Tretinoin, Vandetanib, Vismodegib, Vorinostat, and combinations thereof.

In one embodiment, the second therapeutic agent comprises a smallmolecule multi-kinase inhibitor, e.g., sorafenib or regorafenib. In oneembodiment, the second therapeutic agent comprises a Hedgehog PathwayInhibitor, e.g., vismodegib. In one embodiment, the second therapeuticagent includes a drug selected from Table 2 below.

TABLE 2 Classes Of Drugs Classes of drugs Examples Purine analogsallopurinol, oxypurinol, clofarabine, and tisopurine Pyrimidine5-fluorouracil, Floxuridine (FUDR), capecitabine, analogs cytarabine,6-azauracil (6-AU), and gemcitabine (Gemzar) Proteasome bortezomib,carfilzomib, cecliranib, disulfiram, inhibitorsepigallocatechin-3-gallate, salinosporamide A, ONCX 0912, CEP-18770,MLN9708, epoxomicin, and MG132. Anti- bevacizumab, aflibercept,sunitinib, sorafenib, pazopanib, angiogenic vandetanib, cabozantinib,axitinib, ponatinib, regorafenib, ranibizumab, lapatinib, andvandetanib. Platinum-based cisplatin, carboplatin, oxaliplatin,satraplatin, picoplatin, antineo- nedaplatin, and triplatin. plasticdrugs COX-2 celecoxib, valdecoxib (Bextra), parecoxib (Dynastat),inhibitors lumiracoxib, etoricoxib, and rofecoxib. Nitrogencyclophosphamide, chlorambucil, uramustine, ifosfamide, mustardsmelphalan, bendamustine, and mustine. Alkylating cyclophosphamide,mechlorethamine or mustine (HN2) agents (trade name Mustardgen),uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide,bendamustine, carmustine, lomustine, streptozocin, and busulfan.Anthracyclines Daunorubicin (Daunomycin), Daunorubicin (liposomal),Doxorubicin (Adriamycin), Doxorubicin (liposomal), Epirubicin,Idarubicin, Valrubicin, and Mitoxantrone. Taxanes Paclitaxel (Taxol),Docetaxel (Taxotere), and albumin-bound paclitaxel (Abraxane).Nucleotide methotrexate, pralatrexate, hydroxyurea, and synthesis5-fluorodeoxyuridine, 3,4-dihydroxybenzylamine inhibitor Bcr-ablimatinib, nilotinib, dasatinib, bosutinib and ponatinib. inhibitorsOther arsenic trioxide, thalidomide, revlimid, and mitotane.Topoisomerase amsacrine, etoposide, etoposide phosphate, teniposide,inhibitor doxorubicin, Topotecan (Hycamtin), Irinotecan (CPT-11,Camptosar), Exatecan, Lurtotecan, ST 1481, CKD 602, ICRF-193, andgenistein. HDAC Vorinostat (SAHA), Romidepsin (Istodax), Panobinostatinhibitors (LBH589), Valproic acid (as Mg valproate), Belinostat(PXD101), Mocetinostat (MGCD0103), Abexinostat (PCI-24781), Entinostat(MS-275), SB939, Resminostat (4SC-201), Givinostat, Quisinostat(JNJ-26481585), CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745,ACY-1215, ME-344, sulforaphane, Kevetrin, and ATRA. Multi-kinasesorafenib, regorafenib, and vandetanib. inhibitors Hormone tamoxifen,toremifene, Arimidex (anastrozole), Aromasin therapies (exemestane),Femara (letrozole), and Fulvestrant (Faslodex). Hedgehog vismodegib,BMS-833923, IPI-926, LDE-225, signaling PF-04449913, LEQ 506, andTAK-441. Inhibitors Checkpoint Opdivo (nivolumab), Durvalumab(Medi4736), Keytruda Inhibitors (pembrolizumab, MK3475), BGB-A317,AMP-224, PDR001, REGN 281, Atezolizumab (MPDL3280A), Pidilizumab(BMS-936559, CT-011, ONO-4538), Avelumab (MSB0010718 C), Yervoy(ipilimumab), tremelimumab BCL2 AT-101, Bcl-2/xL inhibitor, Navitoclax(ABT-263), Inhibitors Venetoclax (ABT-199), Apogossypol, PTN1258,obatoclax, G3139

In one embodiment, the second therapeutic agent includes drugs thattarget tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)receptors. In one embodiment, the second therapeutic agent includes arecombinant TRAIL or an agonistic antibody that activates one or moreTRAIL receptors. In one embodiment, the second therapeutic agentincludes one or more antibodies or recombinant TRAIL that activatesignaling by DR4, DR5 or both. In one embodiment, the second therapeuticagent includes one or more of AMG-655, LBY-135, mapatumumab,lexatumumab, Apomab, and rhApo2L/TRAIL. In one embodiment, the secondtherapeutic agent includes an active agent selected from Camptothecin,5-FU, capecitabine, cisplatin, doxorubicin, irinotecan, paclitaxel,cisplatin, bortezomib, BH3I-2, rituximab, radiation, triterpenoids,sorafenib, gemcitabine, HDAC inhibitors, carboplatin, T-101 (a gossypolderivate), ABT-263, ABT-737, and GX-15-070 (obatoclax), vorinostat,cetuximab, panitumumab, bevacizumab, ganitumab, interferon gamma,sorafenib, XIAP antagonists, Bcl-2 antagonists, and Smac mimetics.

VI. DOSE

In one embodiment, a pharmaceutical composition comprises an imipridone,such as ONC201, or an analog thereof, or a pharmaceutically acceptablesalt thereof in a dose ranging from about 40, 50, 60, or 100 mg to about2000 mg; from about 4, 5, 6, or 10 mg to about 200 mg; or from about0.4, 0.5, 0.6, or 1 mg to about 20 mg where the weight can be based onthe compound in its free base form. In one embodiment, a pharmaceuticalcomposition comprises an imipridone, such as ONC201, or an analogthereof, or a pharmaceutically acceptable salt thereof in a dose levelranging from about 50 mg to about 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000mg; from about 5 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, and 200 mg; or from about 0.5 mgto about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,and 20 mg. In one embodiment, a pharmaceutical composition comprises animipridone, such as ONC201, or an analog thereof, or a pharmaceuticallyacceptable salt thereof in a dose level ranging from about 40 mg toabout 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about 4 mg to about20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, or 200 mg; or from about 0.4 mg to about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 mg. In one embodiment,a pharmaceutical composition comprises an imipridone, such as ONC201, oran analog thereof, or a pharmaceutically acceptable salt thereof in adose level ranging from about 60 mg to about 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, or 2000 mg; from about 6 mg to about 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg; or fromabout 0.6 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 mg. In one embodiment, a pharmaceuticalcomposition comprises an imipridone, such as ONC201, or an analogthereof, or a pharmaceutically acceptable salt thereof in a dose levelranging from about 100 mg to about 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 mg, or2000 mg; from about 10 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg; or from about 1mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 mg. In one embodiment, a pharmaceutical composition comprisesan imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof in a dose level ranging fromabout 200 mg to about 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about20 mg to about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, or 200 mg; or from about 2 mg to about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg, based on thecompound in its free base form. In one embodiment, a pharmaceuticalcomposition comprises an imipridone, such as ONC201, or an analogthereof, or a pharmaceutically acceptable salt thereof in a dose levelranging from about 400 mg to about 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about40 mg to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200 mg; or from about 4 mg to about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg based on the compound inits free base form. In one embodiment, a pharmaceutical compositioncomprises an imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof in a dose level ranging fromabout 50 mg to about 60, 70, 80, 90, or 100 mg; from about 60 mg toabout 70, 80, 90, or 100 mg; from about 70 mg to about 80, 90 or 100 mg,from about 80 mg to about 90 or 100 mg; from about 90 mg to about 100mg; from about 5 mg to about 6, 7, 8, 9, or 10 mg; from about 6 mg toabout 7, 8, 9, or 10 mg; from about 7 mg to about 8, 9 or 10 mg, fromabout 8 mg to about 9 or 10 mg; from about 9 mg to about 10 mg; fromabout 0.5 mg to about 0.6, 0.7, 0.8, 0.9, or 1 mg; from about 0.6 mg toabout 0.7, 0.8, 0.9, or 1 mg; from about 0.7 mg to about 0.8, 0.9 or 1mg, from about 0.8 mg to about 0.9 or 1 mg; or from about 0.9 mg toabout 1 mg.

In one embodiment, a pharmaceutical composition comprises an imipridone,such as ONC201, or an analog thereof, or a pharmaceutically acceptablesalt thereof in a dose ranging from about 1 mg/kg to about 40 mg/kg; 0.1mg/kg to about 4 mg/kg; or 0.01 mg/kg to about 0.40 mg/kg. In oneembodiment, a pharmaceutical composition comprises an imipridone, suchas ONC201, or an analog thereof, or a pharmaceutically acceptable saltthereof in a dose level ranging from about 1, 2, 3, 4, 5, 6, 7, 8, or 9mg/kg to about 10, 20, 30, or 40 mg/kg; from about 10, 11, 12, 13, 14,15, 16, 17, 18, or 19 mg/kg to about 20, 30, or 40 mg/kg; from about 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 mg/kg to about 30 or 40 mg/kg;from about 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 mg/kg to about 40mg/kg; from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg/kgto about 1, 2, 3, or 4 mg/kg; from about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, or 1.9 mg/kg to about 2, 3, or 4 mg/kg; from about 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9 mg/kg to about 3 or 4mg/kg; or from about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9mg/kg to about 4 mg/kg; from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,0.07, 0.08, 0.09 mg/kg to about 0.10, 0.20, 0.30, or 0.40 mg/kg; fromabout 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, or 0.19mg/kg to about 0.20, 0.30, or 0.40 mg/kg; from about 0.20, 0.21, 0.22,0.23, 0.24, 0.25, 0.26, 0.27, 0.28, or 0.29 mg/kg to about 0.30 or 0.40mg/kg; or from about 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37,0.38, or 0.39 mg/kg to about 0.40 mg/kg.

In one embodiment, a pharmaceutical composition comprises an imipridone,such as ONC201, or an analog thereof, or a pharmaceutically acceptablesalt thereof in a dose ranging from about 37.5 mg/m² to about 1500mg/m²; from about 3.75 mg/m² to about 150 mg/m²; or from about 0.4 mg/m²to about 15 mg/m² In one embodiment, a pharmaceutical compositioncomprises an imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof in a dose ranging from about40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260,265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400,405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470,475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540,545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610,615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750,755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820,825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890,895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960,965, 970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015, 1020, 1025,1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085,1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145,1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205,1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265,1270, 1275, 1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325,1330, 1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385,1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445,1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495 mg/m² toabout 1500 mg/m²; from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, or 149 mg/m² to about 150 mg/m²; or from about 0.5, 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5,11, 111, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 mg/m² to about 15 mg/m².

VII. DOSAGE FORMS

Suitable pharmaceutical compositions for use in the methods describedherein can be formulated into a dosage form that can be administered toa patient. In one embodiment, a pharmaceutical composition is in theform of an oral dosage unit or parenteral dosage unit. In oneembodiment, a pharmaceutical composition is in the form of an oraldosage unit. In one embodiment, an oral dosage unit is fractionated intoseveral, smaller doses, which are administered to a subject over apredetermined period of time in order to reduce toxicity of atherapeutic agent being administered. In one embodiment, an oral dosageunit is administered by a tablet or capsule comprising a controlledrelease formulation that can include a plurality of particles, granules,pellets, minitablets or tablets. In one embodiment, the pharmaceuticalcomposition is in the form of a parenteral dosage unit. In oneembodiment, the parenteral dosage unit is selected from the groupconsisting of intravenous (IV), subcutaneous (SC), and intramuscular(M), rectal (PR) and transdermal dosage units. In one embodiment, thecomposition is in a dosage form selected from the group consisting ofsterile solutions, suspensions, suppositories, tablets and capsules. Inone embodiment, the composition is an oral dosage form selected from thegroup consisting of a tablet, caplet, capsule, lozenge, syrup, liquid,suspension and elixir. In one embodiment, the composition is in an oraldosage form selected from the group consisting of tablets, hard shellcapsules, soft gelatin capsules, beads, granules, aggregates, powders,gels, solids and semi-solids.

In one embodiment, suitable forms of pharmaceutical compositions for usein the methods described herein include dermatological compositionsadapted for cutaneous topical administration. For example,dermatological compositions include a cosmetically or pharmaceuticallyacceptable medium. Dermatological compositions for topicaladministration can include ointments, lotions, creams, gels, drops,suppositories, sprays, liquids and powders. In one embodiment,conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners, skin enhancers can be necessary or desirable and thereforeused. Examples of suitable enhancers include ethers such as diethyleneglycol monoethyl ether (available commercially as TRANSCUTOL®) anddiethylene glycol monomethyl ether; surfactants such as sodium laurate,sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkoniumchloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80), andlecithin (U.S. Pat. No. 4,783,450); alcohols such as ethanol, propanol,octanol, benzyl alcohol; polyethylene glycol and esters thereof such aspolyethylene glycol monolaurate; amides and other nitrogenous compoundssuch as urea, dimethylacetamide (DMA), dimethylformamide (DMF),2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine; terpenes; alkanones; and organic acids, particularlycitric acid and succinic acid. AZONE® and sulfoxides such as DMSO andCι0MSO may also be used, but are less preferred.

In one embodiment, the pharmaceutical composition is in a dosage formselected from the group consisting of sustained release forms,controlled release forms, delayed release forms and response releaseforms.

VIII. METHODS OF USE

The compositions and methods described herein have utility in treatingmany disease conditions, including cancer (e.g., colorectal, brain, andglioblastoma). In one embodiment, the compositions and methods describedherein are used to treat diseases such as ocular melanoma, desmoplasticround cell tumor, chondrosarcoma, leptomengial disease, diffuse largeB-cell lymphoma, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, AIDS-Related Cancers, AIDS-Related Lymphoma,Anal or Rectal Cancer, Appendix Cancer, Astrocytomas, and AtypicalTeratoid/Rhabdoid Tumor. In one embodiment, the compositions and methodsdescribed herein are used to treat diseases such as Basal CellCarcinoma, Basal Cell Nevus Syndrome, Gorlin-Nevus Syndrome, Bile DuctCancer, Bladder Cancer, Bone Cancer, Osteosarcoma and Malignant FibrousHistiocytoma, Brain Tumor, Breast Cancer, Bronchial Tumors, BurkittLymphoma, and Spinal Cord Tumors. In one embodiment, the compositionsand methods described herein are used to treat diseases such asCarcinoid Tumor, Carcinoma of Unknown Primary, Central Nervous SystemAtypical Teratoid/Rhabdoid Tumor, Leptomeningeal Disease, CentralNervous System Embryonal Tumors, Central Nervous System Lymphoma,Cervical Cancer, Chordoma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Chronic Myeloproliferative Disorders, ColonCancer, Colorectal Cancer, Craniopharyngioma, and Cutaneous T-CellLymphoma (including Sezary syndrome and mycosis fungoides (MF)). In oneembodiment, the compositions and methods described herein are used totreat diseases such as Embryonal Tumors of Central Nervous System,Endometrial Cancer, Ependymoblastoma, Ependymoma, Esophageal Cancer,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, and EyeCancer, including Intraocular Melanoma and Retinoblastoma. In oneembodiment, the compositions and methods described herein are used totreat diseases such as Gallbladder Cancer, Gastric (Stomach) Cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST),Germ Cell Tumor, Gestational Trophoblastic Tumor, and Glioma. In oneembodiment, the compositions and methods described herein are used totreat a cancer selected from the group consisting of Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Hodgkin Lymphoma, and Hypopharyngeal Cancer. In oneembodiment, the compositions and methods described herein are used totreat diseases such as Kaposi Sarcoma and Kidney (Renal Cell) Cancer. Inone embodiment, the compositions and methods described herein are usedto treat diseases such as Langerhans Cell Histiocytosis, LaryngealCancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, includingNon-Small Cell Lung Cancer, and Small Cell Lung Cancer, Non-HodgkinLymphoma, and Primary Central Nervous System Lymphoma. In oneembodiment, the compositions and methods described herein are used totreat diseases such as Waldenström's macroglobulinemia(lymphoplasmacytic lymphoma), Malignant Fibrous Histiocytoma of Bone andOsteosarcoma, Medulloblastoma, Medulloepithelioma, Melanoma, Merkel CellCarcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with OccultPrimary, Multiple Endocrine Neoplasia Syndrome, Mouth Cancer, MultipleMyeloma/Plasma Cell Neoplasm, Mycosis Fungoides, MyelodysplasticSyndromes, complex karyotype, blastic phase leukemia,Myelodysplastic/Myeloproliferative Neoplasms, Multiple Myeloma, andMyeloproliferative Disorders. In one embodiment, the compositions andmethods described herein are used to treat cancer. In one embodiment,the compositions and methods described herein are used to treat diseasessuch as Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,and Neuroblastoma. In one embodiment, the compositions and methodsdescribed herein are used to treat diseases such as Oral Cancer, Lip andOral Cavity Cancer, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer, Ovarian Germ Cell Tumor,Ovarian Epithelial Cancer, and Ovarian Low Malignant Potential Tumor. Inone embodiment, the compositions and methods described herein are usedto treat diseases such as Pancreatic Cancer, Papillomatosis, ParanasalSinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pineal Parenchymal Tumors of IntermediateDifferentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Pleuropulmonary Blastoma,Pregnancy and Breast Cancer, Primary Central Nervous System Lymphoma,and Prostate Cancer. In one embodiment, the compositions and methodsdescribed herein are used to treat a cancer selected from the groupconsisting of Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvisand Ureter, Respiratory Tract Carcinoma Involving the NUT Gene onChromosome 15, Retinoblastoma, and Rhabdomyosarcoma. In one embodiment,the compositions and methods described herein are used to treat highgrade prostate cancer. In one embodiment, the compositions and methodsdescribed herein are used to treat medium grade prostate cancer. In oneembodiment, the compositions and methods described herein are used totreat low grade prostate cancer. In one embodiment, the compositions andmethods described herein are used to treat castration-resistant prostatecancer. In one embodiment, the compositions and methods described hereinare used to treat a nervous system tumor. In one embodiment, thecompositions and methods described herein are used to treat a centralnervous system tumor. In one embodiment, the compositions and methodsdescribed herein are used to treat a peripheral nervous system tumor. Inone embodiment, the compositions and methods described herein are usedto treat a paraganglioma. In one embodiment, the compositions andmethods described herein are used to treat a pheochromocytoma.

In in vitro models, in animal models, and in human clinical trialscompound (1) (ONC201) has broad anti-cancer activity, low toxicityincluding few, if any, adverse effects, low genotoxicity, and highbioavailability including oral bioavailability. These features allowONC201 and various analogs to be particularly well suited for pediatricpatients. These features also make ONC201 and various analogsparticularly well suited for chronic therapy, for high risk patients,and to ensure long-lasting responses or stable disease or to preventdisease recurrence.

In one embodiment, the compositions and methods described herein areused to treat a pediatric cancer (e.g., pediatric solid tumors,pediatric sarcomas, pediatric Ewing's sarcomas, pediatric gliomas,pediatric central nervous system cancers, pediatric neuroblastoma,pediatric leukemia and pediatric lymphoma).

In one embodiment, the compositions and methods described herein areused to treat a proliferative skin disorder such as psoriasis. In oneembodiment, the compositions and methods described herein are used totreat a cancer selected from the group consisting of Salivary GlandCancer, Sarcoma, Sezary Syndrome, Skin Cancer, Ocular Cancer, SkinCarcinoma, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous CellCarcinoma, Squamous Neck Cancer with Occult Primary, and SupratentorialPrimitive Neuroectodermal Tumors. In one embodiment, the compositionsand methods described herein are used to treat a cancer selected fromthe group consisting of T-Cell Lymphoma, Testicular Cancer, ThroatCancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional CellCancer of the Renal Pelvis and Ureter, and Gestational TrophoblasticTumor. In one embodiment, the compositions and methods described hereinare used to treat a cancer selected from the group consisting ofCarcinoma of Unknown Primary Site, Cancer of Unknown Primary Site,Unusual Cancers of Childhood, Transitional Cell Cancer of the RenalPelvis and Ureter, Urethral Cancer, and Uterine Sarcoma. In oneembodiment, the compositions and methods described herein are used totreat cancer selected from the group consisting of Vaginal Cancer andVulvar Cancer. In one embodiment, the compositions and methods describedherein are used to treat a cancer selected from the group consisting ofWilms Tumor and Women's Cancers.

In one embodiment, the compositions and methods described herein areused as a first-line therapy (sometimes called primary therapy). In oneembodiment, the compositions and methods described herein are used as asecond-line therapy. In one embodiment, the compositions and methodsdescribed herein are used as a third-line therapy. In one embodiment,the compositions and methods described herein are used as a salvagetherapy. The term “salvage therapy” means a therapeutic agent that canbe taken with any regimen after a subject's initial treatment regimenhas failed or after the subject's condition has not responded to aninitial treatment. In one embodiment, the compositions and methodsdescribed herein are used as a rescue therapy. In one embodiment of therescue therapy, the compositions are used as a rescue agent tocounteract the action of an initial treatment. In one embodiment of therescue therapy, the compositions are used as rescue agent which isadministered to a subject who has developed resistance to a standard oran initial treatment. In one embodiment, the compositions and methodsdescribed herein are used as a neoadjuvant therapy. In one embodiment,the neoadjuvant therapy comprises administration of one or more of thetherapeutic agents described herein to a subject before a main or firstline treatment. In one embodiment, the neoadjuvant therapy reduces thesize or extent of the cancer being treated before a main or first linetreatment is administered to the subject undergoing treatment. In oneembodiment, the compositions and methods described herein are used as anadjuvant therapy. In one embodiment, the adjuvant therapy comprisesadministration of one or more therapeutic agents described herein to asubject, wherein the one or more therapeutic agent that modify theeffect of other therapeutic agents that are already administered to thesubject or are concurrently administered to the subject or subsequentlyadministered to the subject.

In one embodiment, the compositions and methods described herein exhibitreduced chance of drug-drug interactions. In one embodiment, animipridone, such as ONC201, or an analog thereof are eliminated from thepatient's body before it can interact with another pharmaceuticallyactive agent.

In one embodiment, the compositions and methods of described hereinexhibit toxicity levels that facilitates combinations with otherpharmaceutical agents.

The methods and compositions described herein are not limited to aparticular animal species. In one embodiment, a subject treatedaccording to methods and using compositions described herein, can bemammalian or non-mammalian. In one embodiment, a mammalian subjectmammal includes, but is not limited to, a human; a non-human primate; arodent such as a mouse, rat, or guinea pig; a domesticated pet such as acat or dog; a horse, cow, pig, sheep, goat, or rabbit. In oneembodiment, a non-mammalian subject includes, but is not limited to, abird such as a duck, goose, chicken, or turkey. In one embodiment, thesubject is a human. In one embodiment, subjects can be either gender andany age. The composition and methods can also be used to prevent cancer.The composition and methods can also be used to stimulate the immunesystem.

The methods and compositions described herein are not limited to aparticular age of the subject. In one embodiment, a subject treatedaccording to methods and using compositions described herein is over 50years old, over 55 years old, over 60 years old, or over 65 years old.In one embodiment, a subject treated according to methods and usingcompositions described herein is under 50 years old, under 55 years old,under 60 years old, or under 65 years old.

In one embodiment, a subject treated according to methods and usingcompositions described herein is a pediatric patient. In one embodiment,the pediatric patient is younger than 18 years old, younger than 17years old, younger than 16 years old, younger than 15 years old, youngerthan 14 years old, is younger than 13 years old, younger than 12 yearsold, younger than 11 years old, younger than 10 years old, younger than9 years old, younger than 8 years old, younger than 7 years old, youngerthan 6 years old, younger than 5 years old, younger than 4 years old,younger than 3 years old, younger than 2 years old, younger than 1 yearold. In one embodiment, the pediatric patient is younger than 12 monthsold, younger than 11 months old, younger than 10 months old, youngerthan 9 months old, younger than 8 months old, younger than 7 months old,younger than 6 months old, is younger than 5 months old, younger than 4months old, younger than 3 months old, younger than 2 months old,younger than 1 month old. In one embodiment, the pediatric patientyounger than 4 weeks old, younger than 3 weeks old, younger than 2 weeksold, younger than 1 weeks old. In one embodiment, the pediatric patientis younger than 7 days old, younger than 6 days old, younger than 5 daysold, younger than 4 days old, younger than 3 days old, younger than 2days old, or younger than 1 day old. In one embodiment, the pediatricpatient is a neonate. In one embodiment, the pediatric patient isprematurely born.

In one embodiment, the patient is less than 45 kg in weight, less than40 kg in weight, less than 35 kg in weight, less than 30 kg in weight,less than 25 kg in weight, less than 20 kg in weight, less than 15 kg inweight, less than 14 kg in weight, less than 10 kg in weight, less than5 kg in weight, less than 4 kg in weight, less than 3 kg in weight, lessthan 2 kg in weight, or less than 1 kg in weight.

In one embodiment, the subject has received at least one priortherapeutic agent. In one embodiment the subject has received at leasttwo, at least three, or at least four prior therapeutic agents. In oneembodiment the prior therapeutic agent is ibrutinib, bortezomib,carfilzomib, temozolomide, bevacizumab, cyclophosphamide,hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin,rituximab, 5-fluorouracil, oxaliplatin, leucovorin, or lenalidomide.

In one embodiment, the subject has been treated with radiation. In oneembodiment, the subject has been treated with surgery. In oneembodiment, the subject has been treated with adoptive T-cell therapy.

In one embodiment, the cancer no longer responds to treatment withibrutinib, bortezomib, carfilzomib, temozolomide, bevacizumab,cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone,cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin,leucovorin, lenalidomide, radiation, surgery, or a combination thereof.

In one embodiment, the compositions and methods described herein have adose response relation in cancer cells that is different from the doseresponse relation of the same compositions and methods in normal cells.The dose response relation of ONC201 on proliferation and cell death innormal and tumor cells was determined by measuring cell viabilityfollowing treatment with ONC201 at various concentrations for 72 hours.The tumors tested included a human colon cancer cell line (HCT116),breast tumor cell line (MDA-MB-231), and a human primary glioblastomacell line (U87). And the normal cells tested included human foreskinfibroblasts (HFF), human fetal lung fibroblast (MRC-5) cells, and ahuman lung fibroblast cell line (WI-38). Doxorubicin was used as apositive control at 1 μg/mL in normal fibroblasts. Cell viability ofnormal cells tested was at least about 75% at about 1-5 mg/mL of ONC201,whereas viability of tumor cells was significantly lower (e.g., at orbelow 50%) at the same ONC201 concentration. Moreover, as ONC201concentration increased beyond about 5 mg/mL viability of tumor cellsfell to below 25%, whereas viability of normal cells remained at about75%. Cell viability assays in human fetal lung fibroblast (MRC-5) cellswere performed following 72 hour treatment with compound (1) (5 μM) orDMSO and a recovery period in complete drug-free media after treatment.Cell recovery was seen with ONC201, but not with DMSO.

In one embodiment, the compositions and methods described herein haveutility in treating cancer in a subject. In one embodiment, thecompositions and methods described herein have utility in treatingcancer in a human subject. In one embodiment, the treatment methodcomprises administering to a subject in need of such treatment, apharmaceutically effective amount of an imipridone, such as ONC201, oran analog thereof, or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier.

In one embodiment, the treatment method comprises administering to asubject in need of such treatment: (i) a first therapeutic agentincluding an imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof in combination with (ii) asecond therapeutic agent, wherein the first and the second therapeuticagents are administered either simultaneously or sequentially. Thesecond therapeutic agent can be any suitable therapeutic agent,including any pharmaceutically active agent disclosed herein. Apharmaceutically acceptable ONC201 salt includes the di-hydrochloridesalt below:

It is understood that a di-hydrochloride salt of ONC201 or an analogthereof (including a compound of formula (10)), or an alternativedi-salt thereof apparent from the teaching of this disclosure, can besubstituted for ONC201 or an analog thereof in a composition or dosingregimen described herein.

In one embodiment, the treatment method comprises administering asynergistic pharmaceutical combination, either simultaneously orsequentially, to a subject in need of such treatment, wherein thesynergistic pharmaceutical combination comprises (i) a first therapeuticagent comprising an imipridone, such as ONC201, or an analog thereof, ora pharmaceutically acceptable salt thereof; and (ii) a secondtherapeutic agent. In one embodiment, the treatment method comprisesadministering to a subject in need of such treatment, eithersimultaneously or sequentially, therapeutically synergistic effectiveamounts of the first therapeutic agent in combination with the secondtherapeutic agent. In one embodiment, the treatment method comprisesadministering to a subject in need of such treatment, an effectiveamount of the first therapeutic agent in combination with an effectiveamount of the second therapeutic agent, wherein the combination providesa synergistic effect in the in vivo treatment of a cancer sensitive tothe combination, and wherein the first and the second therapeutic agentsare administered either simultaneously or sequentially. In oneembodiment, the treatment method comprises administering to a subject inneed of such treatment, an effective amount of the first therapeuticagent in combination with an effective amount of a second therapeuticagent, wherein the combination provides a synergistic effect in the invivo treatment of a minimal residual disease sensitive to thecombination, and wherein the first and second therapeutic agents areadministered either simultaneously or sequentially. In one embodiment,the second therapeutic agent is given before or prior to the firsttherapeutic agent.

In one embodiment, the treatment method targets a cancer selected fromthe group consisting of solid tumors, liquid tumors, lymphomas,leukemias, or myelomas.

In one embodiment, the treatment method targets a solid tumor, whereinthe solid tumor is selected from the group consisting of: CervicalCancer, Endometrial Cancer, Extracranial Germ Cell Tumor; ExtragonadalGerm Cell Tumor; Germ Cell Tumor; Gestational Trophoblastic Tumor;Ovarian Cancer, Ovarian Germ Cell Tumor, Ovarian Epithelial Cancer, andOvarian Low Malignant Potential Tumor; Penile Cancer, Prostate Cancer;Pregnancy and Breast Cancer; high grade prostate cancer; medium gradeprostate cancer; low grade prostate cancer; castration-resistantprostate cancer; Breast Cancer; Bile Duct Cancer; Extrahepatic Bile DuctCancer; Gallbladder Cancer; Hepatocellular (Liver) Cancer; Kidney (RenalCell) Cancer; Liver Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis andUreter; Basal Cell Carcinoma; Basal Cell Nevus Syndrome, Gorlin-NevusSyndrome, Melanoma, Merkel Cell Carcinoma, Papillomatosis, MultipleEndocrine Neoplasia Syndrome; Pancreatic Cancer, Parathyroid Cancer,ocular melanoma; Eye Cancer; Retinoblastoma; Malignant FibrousHistiocytoma; Ewing Sarcoma Family of Tumors; desmoplastic round celltumor; chondrosarcoma, Kaposi Sarcoma, Rhabdomyosarcoma; Spinal CordTumors, Leptomeningeal Disease, Central Nervous System Embryonal Tumors,Chordoma, Embryonal Tumors of Central Nervous System, Ependymoblastoma,Ependymoma, Neuroblastoma; Pineal Parenchymal Tumors of IntermediateDifferentiation, Pineoblastoma; Adrenocortical Carcinoma; Bone Cancer,Osteosarcoma; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma;Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; CarcinoidTumor, Carcinoma of Unknown Primary, Bronchial Tumors, Lung Cancer,Pleuropulmonary Blastoma; Respiratory Tract Carcinoma Involving the NUTGene on Chromosome 15, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor;Central Nervous System Atypical Teratoid/Rhabdoid Tumor,Craniopharyngioma, Glioma, Brain cancer, Medulloblastoma,Medulloepithelioma, Supratentorial Primitive Neuroectodermal Tumors;Pituitary Tumor; Gastric (Stomach) Cancer, Gastrointestinal CarcinoidTumor, Gastrointestinal Stromal Tumor (GIST), Bladder Cancer, Anal orRectal Cancer, Appendix Cancer, Esophageal Cancer, HypopharyngealCancer; Laryngeal Cancer, Lip and Oral Cavity Cancer, MetastaticSquamous Neck Cancer with Occult Primary, Mouth Cancer, Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Oral Cancer, Lip and OralCavity Cancer, Oropharyngeal Cancer, Paranasal Sinus and Nasal CavityCancer, Pharyngeal Cancer; Head and Neck Cancer, and Mesothelioma.

In one embodiment, the treatment method targets a lymphoma selected fromthe group consisting of: diffuse large B-cell lymphoma, AIDS-RelatedLymphoma, Cutaneous T-Cell Lymphoma, Sezary syndrome, mycosis fungoides(MF); Histiocytosis; Burkitt Lymphoma, and Central Nervous SystemLymphoma; Non-Hodgkin Lymphoma, and Primary Central Nervous SystemLymphoma, Hodgkin Lymphoma, Waldenström's macroglobulinemia; MycosisFungoides; Primary Central Nervous System Lymphoma; lymphoplasmacyticlymphoma, and Primary Central Nervous System Lymphoma.

In one embodiment, the treatment method targets a Non-Hodgkin's lymphoma(NHL) selected from the group consisting of: mantle cell lymphoma,diffuse large B-cell lymphoma, follicular lymphoma, marginal zonelymphoma, small lymphocytic lymphoma, lyphoplasmacytic NHL,Waldenstrom's macroglobulinemia, and skin lymphomas.

In one embodiment, the treatment method targets a leukemia selected fromthe group consisting of: Acute Lymphoblastic Leukemia (ALL), ChronicLymphocytic Leukemia (CLL), Chronic Myeloproliferative Disorders; HairyCell Leukemia; Acute Myeloid Leukemia (AML); Chronic MyelogenousLeukemia (CML); and Langerhans Cell Histiocytosis.

In one embodiment, the treatment method targets an acute leukemiaselected from the group consisting of: acute lymphocyte leukemia, acutemyeloid leukemia, chronic lymphoblastic leukemia, chronic myeloidleukemia, myelodysplastic syndrome, and myeloproliferative disease.

In one embodiment, the treatment method targets a myeloma selected fromthe group consisting of: IgA myeloma; IgG myeloma; IgM myeloma; IgDmyeloma; IgE myeloma; light chain myeloma; non secretory myeloma;complex karyotype, blastic phase leukemia; Multiple Myeloma/Plasma CellNeoplasm, Multiple Myeloma, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, and MyeloproliferativeDisorders.

In one embodiment, the treatment method targets a peripheral nervoussystem tumor. In one embodiment, the treatment method targets aparaganglioma. In one embodiment, the treatment method targets apheochromocytoma.

In one embodiment, treatment of cancer comprises prevention of tumorgrowth in a cancer subject. In one embodiment, treatment of cancercomprises prevention of formation of cancer metastases in a cancersubject. In one embodiment, treatment of cancer comprises targetedtreatment of minimal residual disease in a cancer subject known to havethe minimal residual disease in a cancer or a subject at risk for havingminimal residual disease.

This might be indicated after treatment of the primary tumor by surgeryand/or after chemotherapy (radiotherapy) has been initiated ordetermined to be efficacious. Disseminated tumor cells may be in theirdormant state and often cannot be attacked by chemotherapy(radiotherapy). A thus treated patient seemingly is in a healed state,and referred to as “minimal residual disease.” Nevertheless, the dormanttumor cells have a potential to form metastases if they becomemetastasizing cells due to a growth stimulus after a longer dormantstate.

The term “minimal residual disease” denotes a small number of cancercells that remain in a subject during or after treatment when thesubject is in remission (exhibiting no symptoms or signs of thedisease). The methods described herein are preferably applied to a formof the diseases listed herein, including adult and childhood forms ofthese diseases.

In one embodiment, the treatment method is useful for treating anautoimmune disease. Autoimmune diseases include, but are not limited toalopecia areata, antiphospholipid, autoimmune hepatitis, celiac disease,diabetes type 1, Graves' disease, Guillain-Barre syndrome, Hashimoto'sdisease, hemolytic anemia, idiopathic thrombocytopenic purpura,inflammatory bowel disease, inflammatory myopathies, multiple sclerosis,primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma,Sjögren's syndrome, systemic lupus erythematosus, and vitiligo.

In one embodiment, the treatment method is useful for treatingautoimmune and inflammatory disorders of the peripheral nerve systemsuch as amyotrophic lateral sclerosis (Lou Gehrig's disease), based onvarious causes such as metabolic disorders that include diabetes, B12and folate vitamin deficiencies, chemotherapy medications and medicinesused to treat HIV, poisons that cause peripheral nerve damage, cancersthat develop peripheral neuropathies as well as paraneoplasticsyndromes, alcohol abuse, chronic kidney disease, injuries that causecompression on nerves and other lesions, infections such as Lymedisease, Guillain Barre syndrome, connective tissue disease, rheumatoidarthritis, Sjogren's syndrome, systemic lupus erythematosus, certaininflammatory conditions such as sarcoidosis, coeliac disease, hereditarydiseases such as charcot marie tooth syndrome, Friedreich's ataxia,and/or idiopathic where no specific cause is found but inflammatoryand/or autoimmune mechanisms are the cause of onset.

In one embodiment, the treatment method is useful for treatingautoimmune and inflammatory disorders with ocular manifestations. Suchocular manifestations include, but are not limited to, ocularcicatricial pemphigoid, Mooren's corneal ulcer, various forms ofuveitis, rheumatoid arthritis, systemic lupus erythematosus,polyarteritis nodosa, relapsing polychondritis, Wegener'sgranulomatosis, scleroderma, Behcet's disease, Reiter's disease,inflammatory bowel disease (ulcerative colitis and Crohn's disease) andankylosing spondylitis, retinitis pigmentosa, macular degeneration,keratoconjunctivitis sicca, scleritis, episcleritis, keratitis,peripheral corneal ulceration, and less common entities such aschoroiditis, retinal vasculitis, episcleral nodules, retinaldetachments, and/or macular edema.

In one embodiment, the treatment method is useful for treating acuteallograft rejection in transplant patients. In one embodiment, thetreatment method is useful for treating ischemic stroke. In oneembodiment, the treatment method is useful for treating inflammatorydiseases including arthritis, psoriasis, asthma, and colitis.

In one embodiment, a therapeutic agent includes a pharmaceuticallyacceptable mono-salt of ONC201 or an analog thereof (e.g., a compound offormula (10)). In one embodiment, a therapeutic agent includes apharmaceutically acceptable ONC201 di-salt or an analog thereof (e.g., acompound of formula (10)). As described herein, some of the analogs canbe tri-salts In one embodiment, a therapeutic agent includes ONC201 oran analog thereof (e.g., a compound of formula (10)) in the form of apharmaceutically acceptable mono- or di-salt selected from the groupconsisting of hydrochloride, hydrobromide, hydrogensulphate, sulfates,phosphates, fumarates, succinates, oxalates and lactates, bisulfates,hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate, malate,maleate, fumarate sulfonate, methylsulfonate, formate, acetate, andcarboxylate. In one embodiment, a therapeutic agent includes ONC201 oran analog thereof in the form of a pharmaceutically acceptable mono- ordi-salt selected from p-toluene-sulfonate, benzenesulfonate,methanesulfonate, oxalate, succinate, tartrate, citrate, fumarate andmaleate. In one embodiment, a therapeutic agent includes ONC201 or ananalog thereof in the form of a pharmaceutically acceptable mono- ordi-salt having a counter ion selected from the group consisting ofammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/orwith counter-ions such as methylamino, dimethylamino, diethylamino,triethylamino counter-ions, and combinations thereof. In one embodiment,a therapeutic agent includes a compound described herein in the form ofa halide di-salt, such as a di-hydrochloride salt or a di-hydrobromidesalt.

In one embodiment of the treatment method, the second therapeutic agentincludes an anti-cancer agent. In one embodiment of the treatmentmethod, the second therapeutic agent is selected from acivicin,aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin,allopurinol, altretamine, ambomycin, ametantrone, amifostine,aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenictrioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin,batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene, bisnafidedimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan,cactinomycin, calusterone, capecitabine, caracemide, carbetimer,carboplatin, carmustine, carubicin, carzelesin, cedefingol, celecoxib,chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate,diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone,duazomycin, edatrexate, elformithine, elsamitrucin, enloplatin,enpromate, epipropidine, epirubicin, erbulozole, esorubicin,estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine,fenretinide, floxuridine, fludarabine, fluorouracil, flurocitabine,fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea,idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2, includingrecombinant interleukin II or rIL2), interferon α-2a, interferon α-2b,interferon α-n1, interferon α-n3, interferon β-Ia, interferon gamma-Ib,iproplatin, irinotecan, lanreotide, letrozole, leuprolide, liarozole,lometrexol, lomustine, losoxantrone, masoprocol, maytansine,mechlorethamine hydrochloride, megestrol, melengestrol acetate,melphalan, menogaril, mercaptopurine, methotrexate, metoprine,meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid,nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel,pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide,pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin,plomestane, porfimer, porfiromycin, prednimustine, procarbazine,puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine,simtrazene, sparfosate, sparsomycin, spirogermanium, spiromustine,spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin,tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide,teroxirone, testolactone, thiamiprine, thioguanine, thiotepa,tiazofurin, tirapazamine, topotecan, toremifene, trestolone,triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard,uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate,vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine,vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate,zorubicin and combinations thereof.

In one embodiment of the treatment method, the second therapeutic agentis selected, from hormone analogs and antihormones, aromataseinhibitors, LHRH agonists and antagonists, inhibitors of growth factors,growth factor antibodies, growth factor receptor antibodies, tyrosinekinase inhibitors; antimetabolites; antitumour antibiotics; platinumderivatives; alkylation agents; antimitotic agents; tubuline inhibitors;PARP inhibitors, topoisomerase inhibitors, serine/threonine kinaseinhibitors, tyrosine kinase inhibitors, protein protein interactioninhibitors, MEK inhibitors, ERK inhibitors, IGF-1R inhibitors, ErbBreceptor inhibitors, rapamycin analogs, amifostin, anagrelid, clodronat,filgrastin, interferon, interferon α, leucovorin, rituximab,procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer,2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyoestradiol,2C4,3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin,16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin,alitretinoin, allovectin-7, altretamine, alvocidib, amonafide,anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, aranose,arglabin, arzoxifene, atamestane, atrasentan, auristatin PE, ABT-199(Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479(ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244(selumetinib), ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717,AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib),avastin, AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006,BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodardicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550,BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid,bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib,brostallicin, busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell,calcitriol, canertinib, canfosfamide, capecitabine,carboxyphthalatoplatin, CC1-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1cefixime, ceflatonin, ceftriaxone, celecoxib, celmoleukin, cemadotin,CH4987655/RO-4987655, chlorotrianisene, cilengitide, ciclosporin,CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicin, combretastatinA4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin52, CTP-37, CTLA-4 monoclonal antibodies, CP-461, CV-247,cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine,deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide,desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol,diflomotecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010,E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFRinhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin,epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3,ethynylcytidine, ethynyloestradiol, exatecan, exatecan mesylate,exemestane, exisulind, fenretinide, figitumumab, floxuridine, folicacid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin,gallium maltolate, gefinitib, gemtuzumab, gimatecan, glufosfamide,GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068,GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100-peptidevaccines, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436(dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183,GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine,histamine, homoharringtonine, hyaluronic acid, hydroxyurea,hydroxyprogesterone caproate, ibandronate, ibritumomab, idatrexate,idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12(cixutumumab), immunol, indisulam, interferon α-2a, interferon α-2b,pegylated interferon α-2b, interleukin-2, INK-1117, INK-128, INSM-18,ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B,isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088,conjugated oestrogens, kahalid F, ketoconazole, KW-2170, KW-2450,lobaplatin, leflunomide, lenograstim, leuprolide, leuporelin,lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol,losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide,marimastat, mechloroethamine, MEK inhibitors, MEK-162,methyltestosteron, methylprednisolone, MEDI-573, MEN-10755, MDX-H210,MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin,mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb,rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453,RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan,R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim,satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101,semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747,SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment of the treatment method, the second therapeutic agentis selected from tamoxifen, toremifene, raloxifene, fulvestrant,megestrol acetate, flutamide, nilutamide, bicalutamide,aminoglutethimide, cyproterone acetate, finasteride, buserelin acetate,fludrocortisone, fluoxymesterone, medroxy-progesterone, octreotide, andcombinations thereof. In one embodiment of the treatment method, thesecond therapeutic agent is selected from LHRH agonists and LHRHantagonists. In one embodiment, a LHRH agonist is selected fromgoserelin acetate, luprolide acetate, triptorelin pamoate andcombinations thereof. In one embodiment, the second therapeutic agentincludes a LHRH antagonist is selected from Degarelix, Cetrorelix,Abarelix, Ozarelix, Degarelix combinations thereof. In one embodiment ofthe treatment method, the second therapeutic agent includes an inhibitorof a growth factor. In one embodiment, the inhibitor of a growth factoris selected from inhibitors of: platelet derived growth factor (PDGF),fibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), insuline-like growth factors(IGF), human epidermal growth factor (HER), hepatocyte growth factor(HGF), and combinations thereof. In one embodiment, the human epidermalgrowth factor (HER) is selected from HER2, HER3, and HER4.

In one embodiment of the treatment method, the second therapeutic agentincludes a tyrosine kinase inhibitor. In one embodiment of the treatmentmethod, the tyrosine kinase inhibitor is selected from cetuximab,gefitinib, imatinib, lapatinib and trastuzumab, and combinationsthereof. In one embodiment of the treatment method, the secondtherapeutic agent includes an aromatase inhibitor. In one embodiment ofthe treatment method, the aromatase inhibitor is selected fromanastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, andcombinations thereof.

In one embodiment of the treatment method, the second therapeutic agentincludes an antimetabolite. In one embodiment of the treatment method,the antimetabolite comprises an antifolate. In one embodiment of thetreatment method, the antifolate is selected from methotrexate,raltitrexed, pyrimidine analogs, and combinations thereof. In oneembodiment of the treatment method, the antimetabolite is a pyrimidineanalog. In one embodiment of the treatment method, the pyrimidine analogis selected from 5-fluorouracil, capecitabin, gemcitabin, andcombination thereof. In one embodiment of the treatment method, theantimetabolite is a purine analog or an adenosine analog. In oneembodiment of the treatment method, the purine analog or adenosineanalog is selected from mercaptopurine, thioguanine, cladribine andpentostatin, cytarabine, fludarabine, and combinations thereof. In oneembodiment of the treatment method, the second therapeutic agentincludes an antitumour antibiotic. In one embodiment of the treatmentmethod, the antitumor antibiotic is selected from anthracyclins,doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C,bleomycin, dactinomycin, plicamycin, streptozocin and combinationsthereof. In one embodiment of the treatment method, the secondtherapeutic agent includes a platinum derivative. In one embodiment ofthe treatment method, the platinum derivative is selected fromcisplatin, oxaliplatin, carboplatin and combinations thereof. In oneembodiment of the treatment method, the second therapeutic agentincludes an alkylation agent. In one embodiment of the treatment method,the alkylation agent is selected from estramustin, meclorethamine,melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide,ifosfamide, temozolomide, nitrosoureas, and combinations thereof. In oneembodiment of the treatment method, the second therapeutic agentincludes a nitrosourea. In one embodiment of the treatment method, thenitrosourea is selected from carmustin, lomustin, thiotepa, andcombinations thereof. In one embodiment of the treatment method, thesecond therapeutic agent includes an antimitotic agent. In oneembodiment of the treatment method, the antimitotic agent is selectedfrom Vinca alkaloids and taxanes. In one embodiment of the treatmentmethod, the taxane is selected from paclitaxel, docetaxel, andcombinations thereof. In one embodiment of the treatment method, theVinca alkaloids are selected from vinblastine, vindesin, vinorelbin,vincristine, and combinations thereof. In one embodiment of thetreatment method, the second therapeutic agent includes a topoisomeraseinhibitor. In one embodiment of the treatment method, the topoisomeraseinhibitor is an epipodophyllotoxin. In one embodiment of the treatmentmethod, the topoisomerase inhibitor, which is an epipodophyllotoxinselected from etoposide, etopophos, teniposide, amsacrin, topotecan,irinotecan, mitoxantron, and combinations thereof. In one embodiment ofthe treatment method, the second therapeutic agent includes aserine/threonine kinase inhibitor. In one embodiment of the treatmentmethod, the serine/threonine kinase inhibitor is selected from PDK 1inhibitors, B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3Kinhibitors, dual mTOR/PI3K inhibitors, STK 33 inhibitors, AKTinhibitors, PLK 1 inhibitors, inhibitors of CDKs, Aurora kinaseinhibitors, and combinations thereof. In one embodiment of the treatmentmethod, the second therapeutic agent includes a tyrosine kinaseinhibitor. In one embodiment of the treatment method, the secondtherapeutic agent includes a PTK2/FAK inhibitor. In one embodiment ofthe treatment method, the second therapeutic agent includes a proteinprotein interaction inhibitor. In one embodiment of the treatmentmethod, the protein protein interaction inhibitor is selected from IAP,Mcl-1, MDM2/MDMX and combinations thereof. In one embodiment of thetreatment method, the second therapeutic agent includes a rapamycinanalog. In one embodiment of the treatment method, the rapamycin analogis selected from everolimus, temsirolimus, ridaforolimus, sirolimus, andcombinations thereof. In one embodiment of the treatment method, thesecond therapeutic agent is selected from amifostin, anagrelid,clodronat, filgrastin, interferon, interferon α, leucovorin, rituximab,procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, andcombinations thereof. In one embodiment of the treatment method, thesecond therapeutic agent is selected from 2-chlorodesoxyadenosine,2-fluorodesoxy-cytidine, 2-methoxyoestradiol, 2C4,3-alethine,131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B,A 105972, A 204197, abiraterone, aldesleukin, alitretinoin,allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole,AG-2037, AP-5280, apaziquone, apomine, aranose, arglabin, arzoxifene,atamestane, atrasentan, auristatin PE, ABT-199 (Venetoclax), ABT-263(Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162,ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (selumetinib),ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055,AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib), avastin,AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate,BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797,BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992(afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI 6727,BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid, bleomycin A,bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin,busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell, calcitriol, canertinib,canfosfamide, capecitabine, carboxyphthalatoplatin, CC1-779, CC-115,CC-223, CEP-701, CEP-751, CBT-1 cefixime, ceflatonin, ceftriaxone,celecoxib, celmoleukin, cemadotin, CH4987655/RO-4987655,chlorotrianisene, cilengitide, ciclosporin, CDA-II, CDC-394, CKD-602,CKI-27, clofarabin, colchicin, combretastatin A4, COT inhibitors,CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin 52, CTP-37, CTLA-4monoclonal antibodies, CP-461, CV-247, cyanomorpholinodoxorubicin,cytarabine, D 24851, decitabine, deoxorubicin, deoxyrubicin,deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone,dexrazoxanet, diethylstilbestrol, diflomotecan, didox, DMDC, dolastatin10, doranidazole, DS-7423, E7010, E-6201, edatrexat, edotreotide,efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509,enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab,ER-86526, erlotinib, ET-18-0CH3, ethynylcytidine, ethynyloestradiol,exatecan, exatecan mesylate, exemestane, exisulind, fenretinide,figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI,formestane, fotemustine, galarubicin, gallium maltolate, gefinitib,gemtuzumab, gimatecan, glufosfamide, GCS-100, GDC-0623, GDC-0941(pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DTimmunogen, GMK, GPX-100, gp100-peptide vaccines, GSK-5126766,GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib),GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795,GW2016, granisetron, herceptine, hexamethylmelamine, histamine,homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesteronecaproate, ibandronate, ibritumomab, idatrexate, idenestrol, IDN-5109,IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), immunol, indisulam,interferon α-2a, interferon α-2b, pegylated interferon α-2b,interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab,iproplatin, irofulven, isohomohalichondrin-B, isoflavone, isotretinoin,ixabepilone, JRX-2, JSF-154, J-107088, conjugated oestrogens, kahalid F,ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim,leuprolide, leuporelin, lexidronam, LGD-1550, linezolid, lutetiumtexaphyrin, lometrexol, losoxantrone, LU 223651, lurtotecan, LY-S6AKT1,LY-2780301, mafosfamide, marimastat, mechloroethamine, MEK inhibitors,MEK-162, methyltestosteron, methylprednisolone, MEDI-573, MEN-10755,MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid,mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb,rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453,RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan,R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim,satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101,semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747,SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment, the other therapeutic agent comprises a steroid,including dexamethasone, prednisolone, methyl prednisolone, prednisone,hydrocortisone, triamcinolone, betamethasone, and cortivazol. In oneembodiment, the other therapeutic agent comprises an anti-emetic.Anti-emetics include, but are not limited to, 5-HT3 receptor agonists(such as dolasetron, granisetron, ondansetron, tropisetron,palonosetron, and mirtazapine), dopamine agonists (such as domperidone,olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine,alizapride, prochlorperazine, and metoclopramide), NK1 receptorantagonists (such as aprepitant and casopitant), antihistamines (such ascyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine,promethazine, hydroxyzine), cannabinoids (such as cannabis, dronabinol,nabilone, and sativex), benzodiazepines (such as midazolam andlorazepam), anticholinergics (such as hyoscine), trimethobenzamide,ginger, emetrol, propofol, peppermint, muscimol, and ajwain.

Pharmaceutical compositions may be administered to a subject via anysuitable route of administration. In one embodiment, the pharmaceuticalcomposition is administered to a subject orally, parenterally,transdermally or transmucosally. In one embodiment, the pharmaceuticalcomposition is administered to a subject parenterally. In oneembodiment, the pharmaceutical composition is administered to a subjectvia a parenteral route of administration selected from the groupconsisting of intravenous (IV), subcutaneous (SC), and intramuscular(IM). In one embodiment, the pharmaceutical composition is administeredto a subject via a route of administration selected from rectal andtransdermal. In one embodiment, the pharmaceutical composition isadministered to a subject in a dosage form selected from the groupconsisting of sterile solutions, suspensions, suppositories, tablets andcapsules. In one embodiment, the pharmaceutical composition isadministered to a subject in an oral dosage form selected from the groupconsisting of a tablet, caplet, capsule, lozenge, syrup, liquid,suspension and elixir. In one embodiment, the pharmaceutical compositionis administered to a subject in an oral dosage form selected from thegroup consisting of tablets, hard shell capsules, soft gelatin capsules,beads, granules, aggregates, powders, gels, solids and semi-solids.

In one embodiment, the pharmaceutical composition is administered to asubject as a dosage form selected from the group consisting of sustainedrelease forms, controlled release forms, delayed release forms andresponse release forms.

In one embodiment, the pharmaceutical composition is administered to asubject once daily. In one embodiment, the pharmaceutical composition isadministered to a subject according to an infrequent dosing regimen(e.g., administered once per week or less frequently). In oneembodiment, the pharmaceutical composition is administered to a subjectaccording to a frequent dosing regimen (e.g., administered more thanonce per week). In one embodiment, the pharmaceutical composition isadministered to a subject once weekly. In one embodiment, thepharmaceutical composition is administered to a subject once every fourweeks. In one embodiment, the pharmaceutical composition is administeredto a subject twice a week. In one embodiment, the pharmaceuticalcomposition is administered to a subject once every two weeks. In oneembodiment, the pharmaceutical composition is administered to a subjectonce every three weeks. In one embodiment, the pharmaceuticalcomposition is administered to a subject in a repeated cycle of onceweekly, once every two weeks, once every three weeks, once every fourweeks or combinations thereof.

In one embodiment, the treatment method comprises administering to asubject in need of such treatment: (i) a first therapeutic agentincluding a compound comprising an imipridone, such as ONC201, or ananalog thereof, or a pharmaceutically acceptable salt thereof incombination with (ii) a second therapeutic agent, wherein the firsttherapeutic agent and the second therapeutic agent are administeredeither simultaneously or sequentially; and further comprises assayingthe expression of an endoplasmic reticulum (ER) stress response gene ina biological sample. In one embodiment, the endoplasmic reticulum stressresponse gene is selected from the group that includes, but is notlimited to, C/EBP-Homologous Protein (CHOP), Activating TranscriptionFactor 3 (ATF3) and both CHOP and ATF3. In one embodiment, theendoplasmic reticulum stress response gene is selected from the groupthat includes, but is not limited to, ATF3, Activating TranscriptionFactor 4 (ATF4) CHOP, IRE1, Binding immunoglobulin protein (BiP),Eukaryotic translation initiation factor 2A (eIF2a), X-box bindingprotein 1 (XBP1). The biological sample may be tumor, peripheral bloodmononuclear cells, or skin biopsy. The biological sample may be obtainedbefore, during, or after drug administration. In one embodiment, thetreatment method further comprises adjusting a dose of the firsttherapeutic agent to achieve induction of about 50%, 75%, 100%, 125%,150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%,450%, 475%, 500%, 525%, 550%, 575%, 600%, or greater than 600% of one ormore ER stress gene. In one embodiment, the treatment method furthercomprises adjusting a dose of the first therapeutic agent to achieveinduction of about 50% to about 100%, about 100% to about 150%, about150% to about 200%, about 200% to about 250%, about 250% to about 300%,about 300% to about 350%, about 350% to about 400%, about 400% to about450%, about 450% to about 500%, about 500% to about 550%, about 550% toabout 600%, or greater than 600% of ER stress genes. In one embodiment,the treatment method further comprises adjusting a dose of the firsttherapeutic agent to achieve induction of about 50% to about 100%, about100% to about 200%, about 200% to about 300%, about 300% to about 400%,about 400% to about 500%, about 500% to about 600%, or greater than 600%of ER stress genes.

In one embodiment, the treatment method comprises administering to asubject in need of such treatment: (i) a first therapeutic agentincluding a compound comprising an imipridone, such as ONC201, an analogthereof, or a pharmaceutically acceptable salt thereof in combinationwith (ii) a second therapeutic agent, wherein the first therapeuticagent and the second therapeutic agent are administered eithersimultaneously or sequentially; and further comprises assaying theexpression of proteasomal activity in a biological sample. In oneembodiment the proteasomal activity may be chymotrypsin-like,trypsin-like, and/or caspase-like activity. In one embodiment, thebiological sample may be tumor, peripheral blood mononuclear cells, orskin cells. The biological sample may be obtained before, during, orafter drug administration. In one embodiment, the treatment methodfurther comprises adjusting the dose to achieve inhibition of about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, or about 100% of the proteasomal activity. In oneembodiment, the treatment method further comprises adjusting the dose toachieve inhibition of at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the proteasomal activity. In oneembodiment, the treatment method further comprises adjusting the dose toachieve inhibition of about 20% to about 30%, about 30% to about 40%,about 40% to about 50%, about 50% to about 60%, about 60% to about 70%,about 70% to about 80%, about 80% to about 90%, or greater than 90% ofthe proteasomal activity.

In an aspect, provided herein are treatment methods, which compriseadministering to a subject in need of such treatment a combination of afirst therapeutic agent including an imipridone, such as ONC201, ananalog thereof, or a pharmaceutically acceptable salt thereof (e.g., adi-salt or tri-salt) and a second therapeutic agent, the methodcomprising:

(i) administering to the subject the first therapeutic agent;

(ii) waiting until a predetermined waiting time has elapsed after thetime of administration of the first therapeutic agent to the subject;and/or until adverse events are resolved or resolving; and

(iii) administering the second therapeutic agent to the subject, whereinthe predetermined waiting time is chosen so as to obtain a delayedtherapeutic effect of the first therapeutic agent without an increasedrisk of possible combined toxic effects of the first and secondtherapeutic agents. In one embodiment, the predetermined waiting time isdetermined based on the clearance rate of the compound of the firsttherapeutic agent or a metabolite thereof. In one embodiment, thepredetermined waiting time is determined by a quantitative assessment ofrenal function and parameters of renal. In one embodiment, thepredetermined waiting time is determined by an assay for thedetermination of renal function, wherein the assay is selected from thegroup consisting of serum level the compound of the first therapeuticagent or a metabolite thereof; clearance rate of the compound of thefirst therapeutic agent or a metabolite thereof; 24-hour urinaryclearance of the compound of the first therapeutic agent or a metabolitethereof.

In one embodiment of the treatment method, the predetermined waitingtime substantially equals the time required for systemic clearance ofthe compound of the first therapeutic agent or a metabolite thereof fromthe subject's body. In one embodiment of the treatment method, thepredetermined waiting time substantially equals the time required forrenal clearance of the compound of the first therapeutic agent or ametabolite thereof from the subject's body. In one embodiment of thetreatment method, the predetermined waiting time substantially equalsthe time required for hepatic clearance of the compound of the firsttherapeutic agent or a metabolite thereof from the subject's body. Inone embodiment of the treatment method, the predetermined waiting timesubstantially equals the time required for total clearance of thecompound of the first therapeutic agent or a metabolite thereof from thesubject's body. In one embodiment of the treatment method, thepredetermined waiting time is about 4 hours. In other embodiments thewaiting time is 1 day. In one embodiment, the waiting time is untilC_(max) of the compound of the first therapeutic agent has passed. Inother embodiments, the waiting time is after most of the adverse eventsare resolved or are resolving. In one embodiment of the treatmentmethod, the predetermined waiting time is about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, or about 7 days. In oneembodiment of the treatment method, the predetermined waiting time is arange of about 1-7 days, about 1-6 days, about 1-5 days, about 1-4 days,about 1-3 days, or about 1 to 2 days. In one embodiment, the waitingtime is up to 3 weeks. The preceeding are considered “therapeutic timeperiods.”

When the order of administration is reversed, timing for theadministration of the first therapeutic agent can be after the C_(max)of the second therapeutic agent (i.e., the first administered drug) haspassed. In one embodiment, administration of the first therapeutic agentcan be after most or substantially all of the first administered drughas been eliminated from the body or the toxicity effects for the firstadministered drug are resolved or are resolving.

In one embodiment, the treatment method further comprises monitoringlevels of the compound of the first therapeutic agent or a metabolitethereof in the subject using pharmacokinetic profiling. In some suchembodiments, monitoring levels of the compound of the first therapeuticagent or a metabolite thereof in the subject using pharmacokineticprofiling comprises constructing a pharmacokinetic profile of thecompound of the first therapeutic agent or a metabolite thereof for thesubject using concentrations of the compound of the first therapeuticagent or a metabolite thereof in at least two samples obtained from thesubject at time points suitable to construct a pharmacokinetic profile.In one embodiment, which include monitoring levels of the compound ofthe first therapeutic agent or a metabolite thereof in the subject usingpharmacokinetic profiling, samples are collected from the subject atpoint-of-care or point of use by sampling or self-sampling onpoint-of-care devices or point of use devices or on matrices suitablefor storage of the samples prior to quantitation in a laboratory. In oneembodiment, each of the point-of-care devices or point of use devices iscapable of quantitating the compound of the first therapeutic agent or ametabolite thereof. In one embodiment, which include monitoring levelsof the compound of the first therapeutic agent or a metabolite thereofin the subject, one or more samples are collected from the subject atpoint-of-care or point of use by biopsy device for analysis at thepoint-of-care or point of use devices or for storage prior to analysisby a laboratory. In one embodiment, a biopsy is taken after a timeinterval of 3-8 hours following administration the first therapeuticagent to the subject. In one embodiment, a biopsy is taken after a timeinterval of 3-24 hours following administration of the first therapeuticagent to the subject. In one embodiment, a biopsy is taken after a timeinterval of 8-24 hours following administration of the first therapeuticagent thereof to the subject. In one embodiment, a biopsy is taken aftera time interval of 2 days following administration of the firsttherapeutic agent to the subject. In one embodiment, a biopsy is takenafter a time interval of 3 days following administration of the firsttherapeutic agent to the subject. In one embodiment, a biopsy is takenafter a time interval of 4 days following administration of the firsttherapeutic agent to the subject. In one embodiment, a biopsy is takenafter a time interval of 1-7 days following administration of the firsttherapeutic agent.

In one embodiment, the pharmacokinetic profile includes pharmacokineticparameters suitable for guiding dosing of the first therapeutic agentfor the subject being treated. In one embodiment of the treatmentmethod, the C_(max) of the first therapeutic agent following itsadministration to the subject ranges from about 1000 ng/dL to 1500 ng/dLfor a therapeutic time period. In one embodiment, C_(max) is less than1500 ng/dL and greater than 85 ng/dL for a therapeutic time period. Inone embodiment, the C_(max) of the first therapeutic following itsadministration to the subject ranges from about 1000 ng/mL to 1500 ng/mLfor a therapeutic time period. In one embodiment, C_(max) is less than1500 ng/mL and greater than 85 ng/mL for a therapeutic time period.

In one embodiment, maximum concentration of the first therapeutic agentin blood (whole blood, plasma, or serum) (“C_(max)”) of a subject afteradministering it to the subject is a C_(max) of from about 1000, 1010,1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130,1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250,1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370,1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or1490 ng/dL to about 1500 ng/dL; from about 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, or 149 ng/dL to about 150 ng/dL; or from about 10, 10.5, 11, 11.5,120, 12.5, 13, 13.5, 14, or 14.5 ng/dL to about 15 ng/dL.

In one embodiment, maximum concentration of the first therapeutic agentin blood (whole blood, plasma, or serum) (“C_(max)”) of the subjectfollowing its administration is a C_(max) of from about 1000, 1010,1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130,1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250,1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370,1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or1490 ng/mL to about 1500 ng/mL; from about 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, or 149 ng/mL to about 150 ng/mL; or from about 10, 10.5, 11, 11.5,120, 12.5, 13, 13.5, 14, or 14.5 ng/mL to about 15 ng/mL.

In one embodiment, maximum concentration of the first therapeutic agentin blood (whole blood, plasma, or serum) (“C_(max)”) of a subjectfollowing its administration is selected from about 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260,1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380,1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or 1490ng/dL. In one embodiment, the C_(max) of the first therapeutic agent inblood (whole blood, plasma, or serum) (“C_(max)”) of a subject followingits administration is selected from about 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, or 149 ng/dL. In one embodiment, the C_(max) of the firsttherapeutic agent following its administration is selected from about10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng/dL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration is selected from about 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160,1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280,1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400,1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or 1490 ng/mL. In oneembodiment, the C_(max) of the first therapeutic agent following itsadministration is selected from about 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or149 ng/mL. In one embodiment, the C_(max) of the first therapeutic agentfollowing its administration is selected from about 10, 10.5, 11, 11.5,120, 12.5, 13, 13.5, 14, or 14.5 ng/mL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration is selected from about 85, 95, 105, 115, 125, 135,145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275,285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415,425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555,565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695,705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835,845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975,985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095,1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215,1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335,1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455,1465, 1475, 1485, 1495, or 1500 ng/dL. In one embodiment, the C_(max) ofthe first therapeutic agent following its administration is selectedfrom about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL. In oneembodiment, the C_(max) of the first therapeutic agent following itsadministration is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, or 14.5 ng/dL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration is selected from about 85, 95, 105, 115, 125, 135,145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275,285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415,425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555,565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695,705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835,845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975,985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095,1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215,1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335,1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455,1465, 1475, 1485, 1495, or 1500 ng/mL. In one embodiment, the C_(max) ofthe first therapeutic following its administration is selected fromabout 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/mL. In oneembodiment, the C_(max) of the first therapeutic agent following itsadministration is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, or 14.5 ng/mL.

In one embodiment, the C_(max) of the first therapeutic agent afteradministering it to the subject ranges from about 85 ng/dL to 1500ng/dL; from about 8.5 ng/dL to 150 ng/dL; or from about 0.85 ng/dL to 15ng/dL. In one embodiment, the C_(max) of the first therapeutic agent ina subject's blood (whole blood, plasma, or serum) after itsadministration is selected from about 85, 95, 105, 115, 125, 135, 145,155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285,295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425,435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565,575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705,715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845,855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985,995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105,1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225,1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345,1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465,1475, 1485, or 1495 ng/dL to about 1500 ng/dL; from about 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, or 149 ng/dL to about 150 ng/dL; or fromabout 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/dL to about15 ng/dL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration ranges from about 85 ng/mL to 1500 ng/mL; from about8.5 ng/mL to 150 ng/mL; or from about 0.85 ng/mL to 15 ng/mL. In oneembodiment, the C_(max) of the first therapeutic following itsadministration is selected from about 85, 95, 105, 115, 125, 135, 145,155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285,295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425,435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565,575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705,715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845,855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985,995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105,1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225,1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345,1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465,1475, 1485, or 1495 ng/mL to about 1500 ng/mL; from about 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, or 149 ng/mL to about 150 ng/mL; or fromabout 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/mL to about15 ng/mL.

In one embodiment, the total drug exposure over time, measured as thearea under the curve (“AUC”) of a plot of the concentration of the drugin blood (whole blood, plasma, or serum) of a subject followingadministration of the drug against time after administration of the drugranges from about 150 ng hr/mL to about 8000 ng hr/mL; from about 15 nghr/mL to about 800 ng hr/mL; or from about 1.5 ng hr/mL to about 80 nghr/mL. In one embodiment, AUC is less than 8000 ng hr/mL and is greaterthan or equal to 150 ng hr/mL. In one embodiment, AUC is less than 800ng hr/mL and is greater than or equal to 15 ng hr/mL. In one embodiment,AUC is less than 80 ng hr/mL and is greater than or equal to 1.5 nghr/mL.

In one embodiment, the total drug exposure over time is an AUC of fromabout 100 ng hr/mL to about 8000 ng hr/mL; from about 10 ng hr/mL toabout 800 ng hr/mL; or from about 1 ng hr/mL to about 80 ng hr/mL. Inone embodiment, the total drug exposure over time is an AUC of fromabout from about 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800,2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600,6800, 7000, 7200, 7400, 7600, or 7800 ng hr/mL to about 8000 ng hr/mL.In one embodiment, the total drug exposure over time is an AUC of fromabout 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540,560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, or 780 ng hr/mLto about 800 ng hr/mL. In one embodiment, the total drug exposure overtime is an AUC of from about from about 1.5, 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78 ng hr/mL to about80 ng hr/mL.

In one embodiment, the total drug exposure over time is an AUC of fromabout 100 ng hr/mL to about 8000 ng hr/mL, from about 10 ng hr/mL toabout 800 ng hr/mL; or from about 1 ng hr/mL to about 80 ng hr/mL. Inone embodiment, the total drug exposure over time is an AUC of fromabout from about 150 ng hr/mL to about 7800, 7600, 7400, 7200, 7000,6800, 6600, 6400, 6200, 6000, 5800, 5600, 5400, 5200, 5000, 4800, 4600,4400, 4200, 4000, 3800, 3600, 3400, 3200, 3000, 2800, 2600, 2400, 2200,2000, 1800, 1600, 1400, 1200, 1000, 800, 600, 400, or 200 ng hr/mL. Inone embodiment, the total drug exposure over time is an AUC of fromabout from about 15 ng hr/mL to about 780, 760, 740, 720, 700, 680, 660,640, 620, 600, 580, 560, 540, 520, 500, 480, 460, 440, 420, 400, 380,360, 340, 320, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100,80, 60, 40, or 20 ng hr/mL. In one embodiment, the total drug exposureover time is an AUC of from about from about 1.5 ng hr/mL to about 78,76, 74, 72, 70, 68, 66, 64, 62, 60, 58, 56, 54, 52, 50, 48, 46, 44, 42,40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4,or 2 ng hr/mL. In one embodiment, the total drug exposure over time isan AUC of from about 100 ng hr/mL to about 200 ng hr/mL; from about 10ng hr/mL to about 20 ng hr/mL; or from about 1 ng hr/mL to about 2 nghr/mL.

In one embodiment, the total drug exposure over time is an AUC selectedfrom about 100, 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800,2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 46000, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600,6800, 7000, 7200, 7400, 7600, 7800, and 8000 ng hr/mL. In oneembodiment, the total drug exposure over time is an AUC selected fromabout 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240,260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 4600, 480, 500, 520,540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, and 800ng hr/mL. In one embodiment, the total drug exposure over time is an AUCselected from about 1, 15, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 460, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, and 80 ng hr/mL.

In another aspect, provided herein are methods of treatment, or use of acomposition to treat a disease state, which comprises administering to asubject in need of such treatment a combination of a first therapeuticagent and a second therapeutic agent, the method comprising:

(i) administering to the subject the first therapeutic agent includingan imipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof;

(ii) monitoring levels of the compound of the first therapeutic agent ora metabolite thereof in the subject using pharmacokinetic profiling; and

(iii) administering the second therapeutic agent conditional on thelevel of the first therapeutic agent in the subject. In one embodiment,the monitoring step includes constructing a pharmacokinetic profile ofthe compound of the first therapeutic agent or a metabolite thereof forthe subject using concentrations of the compound of the firsttherapeutic agent or a metabolite thereof in a plurality of samplesobtained from the subject at time points suitable to construct apharmacokinetic profile. In one embodiment, at least two samples arecollected at point-of-care or point of use by sampling or self-samplingon point-of-care devices or point of use devices or on matrices suitablefor storage of the samples prior to quantitation of the compound or ametabolite thereof by a laboratory. In one embodiment, eachpoint-of-care devices or point of use devices is capable of quantitatingthe compound or a metabolite thereof. In one embodiment, thepharmacokinetic profile includes pharmacokinetic parameters suitable forguiding dosing of the compound or a salt thereof for the subject. In oneembodiment, the samples include from 2-12 samples. In one embodiment,the samples are collected over a time period of up to 8 hours, up to 24hours, up to 48 hours, or up to 72 hours. In one embodiment, thepharmacokinetic parameters include at least one parameter selected fromthe group consisting of AUC, AUC_(inf), T_(max), C_(max), time abovethreshold, steady state concentration, absorption rate, clearance rate,distribution rate, terminal T-½ or parameters drawn fromnoncompartmental pharmacokinetic (PK) or compartmental PK analysis,including physiological model-based compartmental PK analysis. In oneembodiment, the treatment method further comprises generating a reportincluding the pharmacokinetic profile of the subject. In one embodiment,the report includes a recommendation regarding dosing based on thepharmacokinetic profile of the subject. In one embodiment, a reductionin dosage of ONC201, the analog thereof, or the pharmaceuticallyacceptable salt thereof is indicated to reduce risk of toxicity based onone or more pharmacokinetic parameters. In one embodiment, the reductionin dosage of the compound or salt thereof is indicated based on timeabove threshold, wherein the threshold is the drug concentration abovewhich toxicity occurs, or one or more of AUC, AUC_(inf), mean residencetime (MRT), exponentials defining the pharmacokinetic profile, volume ofdistribution at steady state (Vss), volume of distribution during theterminal phase (Vz) or combination of a group of pharmacokineticvariable to adequately describe the pharmacokinetic profile. In oneembodiment, a dose adjustment of the compound or salt thereof isindicated to increase efficacy based on one or more pharmacokineticparameters. In one embodiment, an increase in dosage of the compound orsalt thereof is indicated based on one or more of AUC, AUC_(inf), MRT,exponentials defining the pharmacokinetic profile, steady state volume(Vss) of distribution, volume of distribution during the terminal phase(Vz) or combination of a group of pharmacokinetic variables toadequately describe the pharmacokinetic profile. In one embodiment, thedose of the compound or salt thereof is adjusted to within 5% to 25% ofa desired target value. In one embodiment, each of the samples isapplied to the point-of-care device or the point of use device fordetermining the concentration of the compound or a metabolite thereof,wherein the point-of-care device or the point of use device comprises alateral flow strip having a construction and composition such that anapplication of one or more of the samples to the lateral flow stripcauses a fraction of the drug in the sample to bind to with a componentof the lateral flow strip such that a detectable signal proportional tothe concentration of the drug in the applied sample is produced. In oneembodiment, the samples are applied to matrices suitable for storage ofthe samples prior to quantitation by a laboratory. In one embodiment,the samples are stored as dried blood spots. In one embodiment, drugconcentrations are measured by ELISA, LC MS MS, LC UV or LCMS. In oneembodiment, the pharmacokinetic parameters include at least one ofsteady state concentration, absorption, and terminal T_(1/2). In oneembodiment, at least one of the samples is whole blood.

IX. MULTIMODAL THERAPEUTIC METHODS

In one aspect, provided herein are multimodal therapeutic methods inwhich administration of an imipridone, such as ONC201, an analogthereof, or a pharmaceutically acceptable salt thereof to a subject inneed of such treatment is supplemented by administration of othertherapeutic modalities. In one embodiment, the multimodal therapeuticmethod comprises administering to a subject a pharmaceutical compositioncomprising an imipridone, such as ONC201, an analog thereof, or apharmaceutically acceptable salt thereof in conjunction with radiationtherapy or after radiation is determined to not have been efficacious.In one embodiment, the multimodal therapeutic method comprisesadministering to a subject a pharmaceutical composition comprising animipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof in conjunction with radiation therapy, whereinthe pharmaceutical composition comprising theimipridone, such as ONC201,the analog thereof, or pharmaceutically acceptable salt thereof and theradiation therapy are administered concurrently or sequentially in anyorder. In one embodiment, the multimodal therapeutic method comprisesadministering to a subject a pharmaceutical composition comprising animipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof in conjunction with radiation therapy in asequential arrangement. In one embodiment, the multimodal therapeuticmethod comprises administering to a subject in need of such treatment apharmaceutical composition comprising an imipridone, such as ONC201, ananalog thereof, or a pharmaceutically acceptable salt thereofconcurrently with radiation therapy. In one embodiment, the multimodaltherapeutic method is used for the treatment of cancer. In oneembodiment, the multimodal therapeutic method includes administering toa cancer subject in need of such treatment a pharmaceutical compositioncomprising an imipridone, such as ONC201, an analog thereof, or apharmaceutically acceptable salt thereof and irradiating cancer cellswith a radiation beam. In one embodiment, the multimodal therapeuticmethod uses the technique of conformal radiotherapy (CRT) to deliver adose volume histogram (DVH) prescribed to a cancer subject. In oneembodiment, the multimodal therapeutic method uses the technique ofintensity modulated radiation therapy (IMRT) to deliver radiation tocancer cells. In one embodiment, the multimodal therapeutic method usestechniques that compensate for motion of tumors in the subject duringtreatment (e.g., where doses of radiation must be administered to athoracic tumor which moves as the patient breathes). For example, themultimodal therapeutic method use Four Dimensional Computed Tomography(4D CT) scanning techniques to adjust the delivered radiation field tocompensate for tumor motion over the breathing cycle.

Any suitable type of radiation, including gamma radiation which is givenfractionated, IMRT (intensity modulated radiation therapy), gamma knife,proton therapy and brachytherapy can be used with the multimodaltherapeutic method. Radiation therapy and administering an imipridone,such as ONC201, an analog thereof, or a pharmaceutically acceptable saltthereof can be used to treat brain tumors such as glioblastoma ordisease that has metastasized to the brain from lung cancer. Themultimodal therapeutic method can be used to treat lung cancer,pancreatic cancer, rectal cancer, breast cancer, sarcoma, prostatecancer, gynecological malignancies, and lymphoma. The gamma knife isused frequently to treat brain metastases. In one embodiment, themultimodal therapeutic method includes use of proton therapy to treatcancer, including brain tumors, prostate cancer and any tumor proximatevital organs where it is very important to minimize toxicity to nearbynormal tissue.

In one embodiment, a multimodal therapeutic method includesadministering to a cancer subject in need of such treatment apharmaceutical composition comprising an imipridone, such as ONC201, ananalog thereof, or a pharmaceutically acceptable salt thereof incombination with adoptive cell therapy (e.g., CAR-T (JCAR 14, 15, 16,17, KTE-C19, or CTL019); other T Cell (AFM13); or NK (CDNO-109 orNK-92)) either simultaneously or in combination.

In one embodiment, the multimodal therapeutic method eliminates minimalresidual disease without adding to toxicity resulting from treatment byan imipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof. In one embodiment, the multimodal therapeuticmethod improves prognosis and/or reduces adverse side-effects associatedwith a disease state or condition in a subject undergoing treatment.

X. ADDITIONAL IMIPRIDONE DERIVATIVES, ANALOGS, AND SALTS

In one aspect, provided herein are compounds that are analogs of thecompounds of formula (10) and methods of making them. Persons skilled inthe art will understand that the general principles and conceptsdescribed above in conjunction with ONC201 and compounds of formula (10)and their salts, including principles and concepts related to methodsand pharmaceutical compositions, apply with equal force to the followinganalogs and salts thereof.

In one embodiment, the analogs have the structure of compound (25):

wherein Y is NR₄ or O, and wherein R₁, R₂, R₃, and R₄ independentlyrepresent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl,alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy,aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl,mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl,alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocycle radicals.In one embodiment, R₁, R₂, R₃, and R₄ are optionally substituted. In oneembodiment, some or all hydrogens in R₁, R₂, R₃, and R₄ are substitutedby deuterium. In other embodiments, R₁, R₂, R₃, and R₄ are independentlyselected from the group consisting of H, C₁₋₄ alkyl, C₁₋₄ alkylphenyl,C₁₋₄ alkylphenylketone, C₁₋₄benzyl-piperazine, and C₁₋₄alkylthienyl,wherein C₁₋₄ alkyl, C₁₋₄alkylphenyl, C₁₋₄ alkylphenylketone, andC₁₋₄benzyl-piperazine are optionally substituted with C₁₋₄alkyl,hydroxyl, or halo. In still other embodiments, R₁, R₂, R₃, and R₄ areindependently selected from the group consisting of H, CH₃, CH₂Ph,CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph.

In one embodiment, the analogs have the structure of compound (26):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄ alkyl,C₁₋₄ alkylphenyl, C₁₋₄ alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄ alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionally substitutedwith C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, orhalo. In one embodiment, R₁ is selected from the group consisting of H,CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph.

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X₂₊₁, —OCX₃,or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and where X is ahalogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₂ is a benzyl substituted withone or more of the following substituents alone or in combination in theortho, meta, and/or para positions of the benzyl ring: —CH₃, —NO₂,—OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X₂₊₁,—OCX₃, or —OC_(p)X₂₊₁, where p is an integer from 2 to 20 and where X isa halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenatedC₁₋₄ alkyl, or halo. In one embodiment, the arylalkyl is substitutedwith one or more substituents selected from the group consisting ofhalo, —CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted oran unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl,perhalogenated C₁₋₄ alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (27):

wherein R₁ is H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl,haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl,alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio,alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl,alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocycle radicals.In one embodiment, R₁ is selected from the group consisting of H, C₁₋₄alkyl, C₁₋₄ alkylphenyl, C₁₋₄ alkylphenylketone, C₁₋₄benzyl-piperazine,and C₁₋₄alkylthienyl, wherein C₁₋₄ alkyl, C₁₋₄ alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄ benzyl-piperazine are optionally substitutedwith C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, orhalo. In one embodiment, R₁ is selected from the group consisting of H,CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph.

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₁ is a H. In one embodiment, R₁is a substituted or an unsubstituted arylalkyl, such as a benzyl orphenylethyl group. In one embodiment, the arylalkyl is substituted withC₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, or halo.

In one embodiment, the analogs have the structure of compound (28):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄ alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionally substitutedwith C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, orhalo. In one embodiment, R₁ is selected from the group consisting of H,CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph, CH₂-(2,4-diF-Ph), CH₂-((2-CH₃)-Ph), CH₂CHOHPh, CH₂CH₂(4-N-benzyl-piperazine), and(CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected from the groupconsisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, when R₁ is CH₂Ph, R₂is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ is CH₂-(2,4-diF-Ph). In one embodiment, R₁ is CH₂Ph and R₂ is CH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X₂₊₁, —OCX₃,or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and where X is ahalogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₂ is a benzyl substituted withone or more of the following substituents alone or in combination in theortho, meta, and/or para positions of the benzyl ring: —CH₃, —NO₂,—OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, or —OC_(p)X₂₊₁, where p is an integer from 2 to20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenatedC₁₋₄ alkyl, or halo. In one embodiment, the arylalkyl is substitutedwith one or more substituents selected from the group consisting ofhalo, —CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted oran unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl,perhalogenated C₁₋₄ alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (29):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionally substitutedwith C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, orhalo. In one embodiment, R₁ is selected from the group consisting of H,CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, whenR₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X₂₊₁, —OCX₃,or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and where X is ahalogen including refers to F, Cl, Br, or I; preferably, F, Cl, or Br;more preferably, F or Cl. In one embodiment, R₂ is a benzyl substitutedwith one or more of the following substituents alone or in combinationin the ortho, meta, and/or para positions of the benzyl ring: —CH₃,—NO₂, —OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2to 20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from the group consisting of halo,—CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted or anunsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (30):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄ benzyl-piperazine, and C₁₋₄alkylthienyl, wherein C₁₋₄ alkyl, C₁₋₄ alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄ benzyl-piperazine are optionally substitutedwith C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, orhalo. In one embodiment, R₁ is selected from the group consisting of H,CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, whenR₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including refers to F, Cl, Br, or I, preferably, F, Cl,or Br, more preferably, F or Cl. In one embodiment, R₂ is a benzylsubstituted with one or more of the following substituents alone or incombination in the ortho, meta, and/or para positions of the benzylring: —CH₃, —NO₂, —OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is aninteger from 2 to 20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenatedC₁₋₄ alkyl, or halo. In one embodiment, the arylalkyl is substitutedwith one or more substituents selected from the group consisting ofhalo, —CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted oran unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted orunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (31):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄ alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionally substitutedwith C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenated C₁₋₄ alkyl, orhalo. In one embodiment, R₁ is selected from the group consisting of H,CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, whenR₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X₂₊₁, —OCX₃,or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and where X is ahalogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₂ is a benzyl substituted withone or more of the following substituents alone or in combination in theortho, meta, and/or para positions of the benzyl ring: —CH₃, —NO₂,—OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, or —OC_(p)X₂₊₁, where p is an integer from 2 to20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄ alkoxyl, hydroxyl, perhalogenatedC₁₋₄ alkyl, or halo. In one embodiment, the arylalkyl is substitutedwith one or more substituents selected from the group consisting ofhalo, —CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted oran unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄ alkyl, C₁₋₄ alkoxyl, hydroxyl,perhalogenated C₁₋₄ alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, provided herein are compounds of formula (100):

wherein R₁ and R₂ are independently selected from H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl,aralkoxy, aralkylthio, and acyl radicals. In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph), which is an ONC201 linear isomer (i.e.,TIC-10)

which lacks anti-cancer activity (Jacob et al., Angew. Chem. Int. Ed.,(2014) 53:6628; Wagner et al., Oncotarget (2015) 5(24):12728). But asshown in the Examples TIC-10 is a CXCR7 agonist. CXCR7 agonists can beused for liver regeneration and preventing or treating liver fibrosis(Nature (2014) 505:97).

In one embodiment, R₁ and R₂ are independently selected from the groupconsisting of H, C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone,C₁₋₄benzyl-piperazine, C₁₋₄alkylthienyl, C₁₋₄alkylpyridinyl,C₁₋₄alkylisoxazolidinyl, C₁₋₄alkylmorpholinyl, C₁₋₄alkylthiazolyl, andC₁₋₄alkylpyrazinyl wherein C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄ benzyl-piperazine, C₁₋₄alkylthienyl,C₁₋₄alkylpyridinyl, C₁₋₄ alkylisoxazolidinyl, C₁₋₄ alkylmorpholinyl,C₁₋₄alkylthiazolyl, and C₁₋₄ alkylpyrazinyl are optionally substitutedwith C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, orhalo. In one embodiment, R₁ and/or R₂ is a substituted or unsubstituted,arylalkyl or heteroarylalkyl. In one embodiment, the heteroarylalkyl isselected from C₁₋₄alkylpyrrolyl, C₁₋₄alkylfuryl, C₁₋₄alkylpyridyl,C₁₋₄alkyl-1,2,4-thiadiazolyl, C₁₋₄alkylpyrimidyl, C₁₋₄alkylthienyl, C₁₋₄alkylisothiazolyl, C₁₋₄ alkylimidazolyl, C₁₋₄ alkyltetrazolyl, C₁₋₄alkylpyrazinyl, C₁₋₄ alkylpyrimidyl, C₁₋₄ alkylquinolyl, C₁₋₄alkylisoquinolyl, C₁₋₄ alkylthiophenyl, C₁₋₄ alkylbenzothienyl, C₁₋₄alkylisobenzofuryl, C₁₋₄ alkylpyrazolyl, C₁₋₄alkylindolyl,C₁₋₄alkylpurinyl, C₁₋₄alkylcarbazolyl, C₁₋₄alkylbenzimidazolyl, andC₁₋₄alkylisoxazolyl.

In one embodiment, R₁ and/or R₂ is a benzyl optionally substituted withone or more of the following substituents on the benzyl ring: X, —CH₃,—NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂-C₄ alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1), —OC_(p)X_(2p+1),OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m),C(O)R^(m), and C(O)OR^(m); R^(m) and R^(n) are independently selectedfrom H or a C₁-C₄ alkyl; and where p is an integer from 2 to 20 and X isa halogen, including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl.

XI. EXAMPLES

It should be understood that the description and examples below aremeant for purposes of illustration only and are not meant to limit thescope of this disclosure. The examples below are meant to illustrate theembodiments disclosed and are not to be construed as being limitationsto them. Additional compounds, other than those described below, may beprepared by the following reaction schemes or appropriate variations ormodifications thereof.

Example 1. Synthesis of 2-Chlorobenzylamino-2-imidazoline Hydriodide

To a stirred solution of 2-methylthio-2-imidazoline hydriodide (244 mg,1.00 mMol) in dry dioxane (2.0 mL) was added 2-chlorobenzylamine (141mg, 1.0 mMol). The reaction mixture was stirred for 90 min at 70° C.under argon. The solution was cooled to room temperature, filtered on asintered funnel, washed with cold dioxane (2 mL) and dried under vacuum.The white solid compound 4.HI (R₂=2-chlorobenzyl) was obtained (242 mg,72%) and used without further purification.

Example 2. Synthesis of 2-Chlorobenzylamino-2-imidazoline

To a stirred solution of 2-chlorobenzylamino-2-imidazoline hydriodide(242 mg, 0.72 mMol) in water (3 mL), was added 1.0 N sodium hydroxide (2mL) at 7° C. The reaction mixture was stirred for 30 min at 7° C. underargon. After that methylene chloride (5 mL) was added and the mixturestirred for another 5 min. The reaction mixture was extracted withmethylene chloride (2×2.5 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered and evaporated. The resulting free base (150mg, 100%) was obtained as a viscous liquid and was used for the nextreaction without any further purification. MS(ESI) 210(M+H).

Example 3. Synthesis of Methyl-1-benzyl 4-oxo-3-piperidine Carboxylate(Compound (6)

To a stirred methyl-1-benzyl 4-oxo-3-piperidine carboxylatehydrochloride (5.7 g, 20 mMol) in ethyl acetate (50 mL), was addedtriethylamine (6 mL) at 7° C. The reaction mixture was stirred for 30min at 7° C. under an argon atmosphere. The reaction mixture wasextracted with ethyl acetate (2×50 mL) and washed with water (50 mL).The organic layer was dried over anhydrous Na₂SO₄, filtered andevaporated. The resulting free base residue (5, R₁=benzyl) as a viscousoil was used in the next reaction without any further purificationMS(ESI) 248(M+H)

Example 4. Synthesis of ONC202 (Compound (14)

To a solution of 2-chlorobenzylamino-2-imidazoline (150 mg, 0.72 mMol),methyl 1-benzyl 4-oxo-3-piperidine carboxylate (5, R₁=benzyl) (195 mg,0.79 mMol) in 1-butanol (2 mL) was added PPTS (10 mg) and the mixturewas stirred at room temperature for 48 h. After that the reactionmixture was refluxed at 125° C. to 130° C. for 2 h. The solvents wereremoved under vacuum, extracted with ethyl acetate (10 mL), and washedwith saturated sodium bicarbonate solution (2×10 mL) and water (10 mL).The organic layer was dried over anhydrous Na₂SO₄, filtered andevaporated. The crude free base was purified by RP HPLC (10%-40%acetonitrile/water) to give ONC902 TFA salt as a white solid (228 mg,50% yield) MS(ESI) 407 (M+H).

The same process was used starting with different benzylamines toprepare various analogs, e.g., ONC203, 204, 205, 206, 912, 210, 211,212, 213, 214, 217, 218, 219, 220, 221, 222, 223, 224, 225, and 226.

Example 5. Synthesis of ONC207 (Compound (19)

To a suspension of 60% sodium hydride (3.5 g, 88 mMol) in dry toluene(50 mL), dimethyl carbonate (4.32 g, 48.0 mMol) was added dropwise in0.5 h at room temperature under nitrogen. After addition of a few dropsof methanol, 1-tert-butoxycarbonyl-4-piperidone (4.8 g, 24 mMol)dissolved in dry toluene (20 mL) was added dropwise to the reactionmixture while stirring at 80° C. over 1 h. The reaction mixture wasstirred for 3 h at the same temperature and then cooled to 0° C. (icebath) and adjusted to pH 6-6.5 with acetic acid. The resulting coldmixture was diluted with water (10 mL) and adjusted to pH 8 with 5%sodium hydroxide solution. The toluene layer was separated and theaqueous layer was extracted with toluene (20 mL). The combined organiclayer was dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The compound was dried in vacuum to givemethyl-1-tert-butoxycarbonyl-4-oxo-3-piperidine carboxylate (5.0 g,80%). The compound obtained was carried to the next reaction without anyfurther purification.

2-methylbenzylamino-2-imidazoline (190 mg, 1 mMol), methyl1-tert-butoxycarbonyl-4-oxo-3-piperidine carboxylate (315 mg, 1.1 mMol)in 1-butanol (2 mL) was added PPTS (10.0 mg) and the mixture was stirredat room temperature for 48 h. After that the reaction mixture wasrefluxed at 125° C. to 130° C. for 2 h. The solvents were removed undervacuum, extracted with ethyl acetate (10 mL), washed with saturatedsodium bicarbonate solution (2×10 mL) and water (10 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and evaporated. Thecrude free base was cleaved with 10% trifluoroacetic acid indichloromethane, purified by RP HPLC (10%-40% acetonitrile/water) togive ONC907 (262 mg, 50%) TFA salt as a white solid MS(ESI) 297 (M+H).

Example 6. Synthesis of ONC209 (Compound (21)

A mixture of ONC907 (100 mg, 0.2 mMol), phenylethyl bromide (55.0 mg,0.28 mMol) and potassium carbonate (150 mg, 1.0 mMol) inN,N-dimethylformamide (3 mL) was heated to 70° C. for 12 h. The solventswere removed under vacuum, extracted with ethyl acetate (10 mL), andwashed with water (5 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and evaporated. The crude free base was purified by RPHPLC (10%-40% acetonitrile/water) to give ONC209 (62 mg, 50%) TFA saltas a white solid MS(ESI) 401 (M+H).

The same process was used starting with different halides to give ONC215and 214. Compounds 227, 228, 229, 230, 231, 232, 233, 234, 235, and 236were prepared using an analogous process from Examples 1 and 5 startingwith different benzylamines. Then treating the intermediate compoundwhere R₁ is H with different halides as above.

Compound ONC216 was prepared from ONC215 by treatment with TFA.

Compound (72) was prepared by reacting the precursor NH compoundprepared in analogy to Example 5 and treating it with styrene oxide.

Example 7. Synthesis of ONC208 (Compound (20)

To a solution of 2-methylbenzylamino-2-imidazoline (190.0 mg, 1.0 mmol),methyl 1-methyl 4-oxo-3-piperidine carboxylate (185.0 mg, 1.0 mMol) in1-butanol (2.0 mL) was added PPTS (10.0 mg) and the mixture was stirredat room temperature for 48 h. After that the reaction mixture wasrefluxed at 125° C. to 130° C. for 2 h. The solvents were removed undervacuum, extracted with ethyl acetate (10 mL), washed with saturatedsodium bicarbonate solution (2×10 mL) and water (10 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and evaporated. Thecrude free base was purified by HPLC 10%-40% acetonitrile and water togive ONC908 (270.0 mg, 50%) TFA salt as a white solid MS(ESI) 311 (M+H).

Example 8. Synthesis of ONC201 (Compound (1)

To a stirred 800 mL saturated NaHCO₃ in a 2 L round bottom flask,compound (3) (239.7 g, 0.845 mol, 1.6 equiv) was added in portions.n-Butanol (500 mL) was added to the resulting mixture, which was stirredfor 30 min and then transferred to a separating funnel. The organicphase, containing compound (4), was separated and transferred to a 2 Lthree-neck round bottom flask equipped with mechanical stirring, N₂inlet, a thermocouple, a condenser and a Dean-Stark trap. To thecontents of the flask, Compound (5) (100 g, 0.528 mol, 1 equiv) andpyridinium p-toluenesulfonate (PPTS) (6.63 gm 0.026 mol, 5 mol %) wereadded. The resulting mixture was heated to reflux for 6 hours. Water inthe reaction mixture was separated into the Dean-Stark trap asnecessary. Refluxing temperature increased from 93° C. to 118° C.Reaction progress was monitored by HPLC. When the peak area of compound(1) on HPLC remained constant with the reaction time, the reaction wasstopped.

Example 9. Synthesis of Di-Salt of ONC201 (Compound (1).2HCl

Without isolation of the compound (1), the reaction mixture from Example8 was washed with water (500 mL) and diluted with methyl tert-butylether (MTBE) (800 mL). The organic phase was washed with water (500mL×2) and transferred to a 3 L three-neck round bottom flask equippedwith mechanical stirring, N₂ inlet, a thermocouple, a condenser and aDean-Stark trap. While agitating the reaction mixture, 1 N HCl indioxane-MTBE solution was added dropwise (4 N HCl in dioxane: 300 mL,1.2 mol, 2.27 equiv; MTBE: 1200 mL) until no more solid precipitated outof the reaction mixture upon addition of HCl. The reaction mixture washeated to reflux at 60-65° C. for 2 hours. Water was separated into theDean-Stark trap as necessary. Upon cooling to room temperature, thesolid precipitate was filtered through a sintered glass funnel andwashed with n-butanol-MTBE (1:2, 600 mL) and MTBE (600 mL) respectively.The solid was dried in a vacuum oven at 65° C. overnight (16 hours) toafford 200 g yellow solid.

To a 2 L three-neck round bottom flask equipped with mechanicalstirring, N₂ inlet, a thermocouple and a condenser, the above solid (200g) was added, followed by ethanol (1000 mL). The mixture was heated toreflux at 78° C. for 2 hours. Upon cooling to room temperature, thesolid was filtered through a sintered glass funnel and washed withethanol (200 mL×3). The wet solid was dried in the vacuum oven at 85° C.for 3 days until the residual solvent met specification. 120 g ofcompound (2) was obtained as a white solid in a yield of 49%, with HPLCpurity 99.7%.

Example 10. Activity of Imipridones

A number of imipridones were prepared based on the syntheses above. Foreach compound, viability of human cancer cells at 72 hourspost-treatment with the compound was measured. The change in potency(relative to ONC201) was determined and shown in Table 3.

TABLE 3 RELATIVE POTENCY OF ONC201 ANALOGS Relative No. Identifier R₁ R₂Potency*  1 ONC201 CH₂Ph CH₂-((2-CH₃)—Ph) N/A 14 ONC202 CH₂PhCH₂(2-Cl—Ph) B 15 ONC203 CH₂Ph CH₂-(2-thienyl) C 16 ONC204 CH₂PhCH₂CH₂Ph C 17 ONC205 CH₂Ph CH₂CH₂(4-N-benzyl-piperazine) C 18 ONC206CH₂Ph CH₂-(2,4-di F—Ph) A 19 ONC207 H CH₂-((2-CH₃)—Ph) B 20 ONC208 CH₃CH₂-((2-CH₃)—Ph) B 21 ONC209 CH₂CH₂Ph CH₂-((2-CH₃)—Ph) B 32 ONC215(CH₂)₃—NH—BOC CH₂-((2-CH₃)—Ph) B 33 ONC216 (CH₂)₃—NH₂ CH₂-((2-CH₃)—Ph) B41 ONC210 CH₂Ph CH₂-(3,5-di F—Ph) B 51 ONC211 CH₂Ph CH₂-(3,4-di Cl—Ph) B52 ONC212 CH₂Ph CH₂-(4-CF₃—Ph) A 53 ONC213 CH₂Ph CH₂-(3,4-di F—Ph) A 54ONC214 CD₂C₆D₅ CH₂-((2-CH₃)—Ph) B 43 ONC217 CH₂Ph CH₂(2-F—Ph) C 55ONC218 CH₂Ph CH₂(2-CH₃, 4-F—Ph) A 56 ONC219 CH₂Ph CH₂-(2,4-di Cl—Ph) A57 ONC220 CH₂Ph CH₂-((4-OCH₃)—Ph) A 35 ONC222 CH₂PhCH₂-(3-isoxazolidinyl) C 36 ONC224 CH₂Ph CH₂CH₂-(4-morpholinyl) A 38ONC221 H CH₂-(4-CF₃—Ph) A 72 ONC225 CH₂Ph CH₂-(2-F, 4-CF₃—Ph) A 37ONC223 CH₂Ph CH₂-(4-CH₃—Ph) A 34 ONC226 CH₂Ph CH₂-(3-pyridinyl) A 77ONC231 CH₂-3-pyridyl CH₂-(4-CF₃—Ph) A 78 ONC232 CH₂-4-methyl-2-thiazolylCH₂-(4-CF₃—Ph) B 79 ONC233 CH₂-2-pyrazinyl CH₂-(4-CF₃—Ph) B 81 ONC234CH₂-(3,4-di Cl—Ph) CH₂-(4-CF₃—Ph) A 83 ONC236 CH₂-3-thienylCH₂-(4-CF₃—Ph) A 84 ONC237 CH₂CH(OH)Ph CH₂-(4-CF₃—Ph) C 73 ONC227CH₂-(4-CF₃—Ph) CH₂-(4-CF₃—Ph) B 74 ONC228 CH₂-(4-F—Ph) CH₂-(4-CF₃—Ph) A75 ONC229 CH₂-(4-OCH₃—Ph) CH₂-(4-CF₃—Ph) B 76 0NC230 4-F—Ph-4-oxobutylCH₂-(4-CF₃—Ph) A *Relative to the potency of ONC201; A Indicates apotency increase of >2-fold of ONC201; B Indicates potency that iswithin 2-fold of ONC201; and C Indicates a potency decrease of >2-foldof ONC201.

The IC₅₀ of ONC201 and ONC212 (5 nM-5 μM, 72 h) upon treatment ofseveral acute myeloid leukemia (AML) cell lines (n=3) were determinedand shown below in Table 11.

TABLE 11 AML cell line ONC201 IC₅₀ (μM) ONC212 IC₅₀ (μM) MV411 3.25 0.01HL60 >5 0.21 MOLM14 3.92 0.01

Cell viability of MV411 AML cells treated with ONC212 and cytarabine (5nM-5 μM, 24 h) (n=3) was measured (FIG. 29A). Furthermore, cellviability MOLM14, MV411 AML cells, MRC5 lung fibroblasts and Hs27a bonemarrow cells treated with ONC212 (5 nM-5 μM, 72 h) (n=3) was measured(FIG. 29B). Cell viability of MOLM14 and MV411 AML cells treated withONC212 (250 nM) for 4, 8, 24, 48, 72 and 96 h was measured. ONC212medium was replaced by fresh medium at these time points and cellviability was determine at 96 h for all samples. (n=2) (FIG. 29C).

In addition, a single dose of compound (52) (ONC212) by oral orintraperitoneal administration to human colon cancer xenograft-bearingmice resulted in significant reduction of tumor volume compared tovehicle-treated control cohorts (FIG. 24). Compound (52) has a widetherapeutic window, as it is well tolerated at doses at least up to 225mg/kg in mice.

Furthermore, ONC212 demonstrated efficacy in ONC201-resistant AMLxenograft model (FIG. 30). MV411 AML cells (5×10⁶) were subcutaneouslyimplanted in the flanks of athymic nude. ONC212 and ONC201 wereadministered orally (PO) as indicated. Tumor volume (A and B) and bodyweight (C) (n=10) was measured on indicated days. * represents p<0.05relative to vehicle.

ONC212 efficacy in AML was evaluated in vitro and was up to 400 foldmore potent compared to ONC201 (Table 11). ONC212 was also efficaciousin AML cells resistant to standard of care cytarabine (FIG. 29A).Despite robust improvement in efficacy ONC212 maintains a widetherapeutic window in vitro and is non-toxic to normal cells atefficacious concentrations (FIG. 29B). An 8 hr exposure of ONC212 at 250nM was sufficient to cause robust reduction in cell viability in MOLM14and MV411 AML cells (FIG. 29C). At least 24-48 h exposure was requiredwith ONC201 for efficacy.

ONC212 efficacy was determined in a leukemia xenograft model with MV411AML cells resistant to standard-of-care cytarabine (FIG. 30). ONC212 50mg/kg significantly reduced leukemia xenograft tumor growth with oralweekly administration while ONC201 was not efficacious in this model atsimilar doses (FIG. 30A). Interesting, biweekly ONC212 dosing with 25mg/kg and weekly/biweekly dosing with 5 mg/kg was not efficacious (FIG.30B). None of these ONC212 administration regimens were associated withbody weight loss (FIG. 30C) or gross observations.

ONC212 25 mg/kg represents NOAEL in mouse and rat non-GLP oral singledose studies which is also the efficacious dose in mouse xenograftstudies. ONC212 is approximately 10 fold more toxic compared to ONC201(NOAEL 225 mg/kg in rat non-GLP oral single dose study).

ONC206 demonstrated efficacy in a Ewing's sarcoma xenograft model (FIG.31). MHH-ES-1 Ewing's sarcoma cells (5×10⁶) were subcutaneouslyimplanted in the flanks of athymic nude mice. ONC206 (PO) andmethotrexate (IV) were administered on day 1 and day 13 as indicated.Tumor volume (FIG. 31A) and body weight (FIG. 31B) (n=4) was measured onindicated days.

In addition, the IC₅₀ of ONC201 and ONC206 (5 nM-5 μM, 72 h) upontreatment of several cell lines (n=3) were determined and shown below inTable 11.

TABLE 12 ONC201 ONC206 Cell line IC₅₀ (μM) IC₅₀ (μM) MV411 (AML) 3.250.2 K562 (CML) >5 0.22 MOLM14 (AML) 3.92 0.27 MHH-ES-1 (Ewing's sarcoma)5.65 0.61 HFF (Normal Fibroblast) >5

ONC206 showed up to 20 fold improvement compared to ONC201 in in vitropotency with no in vitro toxicity to normal cells at therapeutic doses(Table 12). With ONC206, only 2-fold increased toxicity (NOAEL 125mg/kg) was noted overall relative to ONC201 (NOAEL 225 mg/kg) in ratnon-GLP oral single dose study. In vivo efficacy in Ewing's sarcomamodel with no toxicity (FIG. 31). ONC206 efficacy was comparable tochemotherapy methotrexate, but chemotherapy was associated with bodyweight loss.

In vitro profiling of GPCR activity using a heterologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC213 selectively targets DRD2/3 and GPR132/91 (FIG. 32). Dualtargeting of DRD2/3 and GPR132/91 represents a novel strategy foranti-cancer efficacy without toxicity. ONC213 is a DRD2/3 inhibitor anda GPR132/91 agonist. DRD2/3 potency of ONC213 is more than ONC201 butless than ONC206. GPR132 potency of ONC213 is less than ONC212.Specifically, ONC213 demonstrated in vitro anti-cancer potency inHCT116/RPMI8226 cancer cells similar to ONC212, but in vitro toxicity tonormal cells was reduced compared to ONC212 (FIG. 33). The safetyprofile of ONC213 confirmed in mouse MTD study with NOAEL 75 mg/kg threetimes that of ONC212 (25 mg/kg). The GPR91 agonist activity of ONC213provides an opportunity for immunology, immune-oncology andhematopoietic applications (Nature Immunology 9:1261 (2008); J LeukocBiol. 85(5):837 (May 2009)).

In vitro profiling of GPCR activity using a heterologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC237 selectively targets DRD5 and GPR132 (FIG. 34). ONC237 is a GPR132agonist and DRD5 antagonist and has reduced anticancer efficacy (IC₅₀31.2 μM) compared to ONC201. This data show that combining GPR132agonist activity with DRD5 (D1-like dopamine receptor) antagonistactivity results in poor anti-cancer effects compared to ONC213 whichcombines GPR132 agonist and DRD2/3 antagonist activity.

In vitro profiling of GPCR activity using a heterologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC236 is a highly selective GPR132 agonist (FIG. 35). ONC236 hasanticancer efficacy (IC₅₀ 88 nM) comparable to ONC212 (10 nM) betterthan ONC206/ONC201, completeness of response is better than ONC201 butnot ONC212 in HCT116 cells.

In vitro profiling of GPCR activity using a heterologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC234 is a broad spectrum and potent GPCR targeting small molecule(FIGS. 36 and 38). ONC234 hits several GPCRs including activity as anantagonist activity for adrenergic, histamine, serotonin, CHRM, CCR,DRD2/5 receptors, as well as CXCR7 agonist activity. ONC236 hasanticancer efficacy (IC₅₀ 234 nM) similar to ONC206, completeness ofresponse same as ONC212, and better than ONC201 in HCT116 cells.

ONC201 Linear Isomer (TIC-10)

In vitro profiling of GPCR activity using a heterologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatthe ONC201 linear isomer (TIC-10) is a CXCR7 agonist (FIG. 37). CXCR7agonists can be used for liver regeneration and preventing/treatingfibrosis, such as liver fibrosis (Nature 505:97 (2014)). Fibrosis is theformation of excess fibrous connective tissue in an organ or tissue,including as a result of wound healing. Examples of fibrosis includes,pulmonary fibrosis, including cystic fibrosis and idiopathic pulmonaryfibrosis; radiation-induced lung injury following treatment for cancer;liver fibrosis (cirrhosis); heart fibrosis, including atrial fibrosis,endomyocardial fibrosis, and old myocardial infarction; glial scar;arthrofibrosis; Crohn's Disease; dupuytren's contracture; keloids;mediastinal fibrosis; myelofibrosis; Peyronie's disease; nephrogenicsystemic fibrosis; progressive massive fibrosis; retroperitonealfibrosis; scleroderma/systemic sclerosis; and adhesive capsulitis.

Example 11. GPCR Antagonism of ONC201

ONC201 was evaluated in a whole cell, functional assay of β-Arrestin Gprotein-coupled receptor (GPCR) activity that directly measures dopaminereceptor activity by detecting the interaction of β-Arrestin with theactivated GPCR that serves as a reporter. For each dopamine receptor(DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5), cell lines overexpressingreporter constructs were expanded from freezer stocks. Cells were seededin a total volume of 20 μL into white walled, 384-well microplates andincubated at 37° C. prior to testing, with antagonist followed byagonist challenge at the EC₈₀ concentration. Intermediate dilution ofsample stocks was performed to generate 5× sample in assay buffer. 3.5μL of 5× sample was added to cells and incubated at 37° C. or roomtemperature for 30 minutes. Vehicle concentration was 1%. 5 μL of 6×EC₈₀agonist in assay buffer was added to cells and incubated at 37° C. orroom temperature for 90 or 180 minutes prior to assay readout. %Antagonism was calculated using the following formula %:Antagonism=100%×(1−(mean RLU of test sample−mean RLU of vehiclecontrol)/(mean RLU of EC₈₀ control−mean RLU of vehicle control).

Example 12: Selective Antagonism of DRD2 by ONC201

ONC201 is a first-in-class small molecule discovered in a phenotypicscreen for p53-independent inducers of tumor selective proapoptoticpathways. Oral ONC201 is being evaluated as a new therapeutic agent infive early phase clinical trials for select advanced cancers based onpronounced efficacy in aggressive and refractory tumors and excellentsafety.

In this Example, the prediction and validation of selective directmolecular interactions between ONC201 and specific dopamine receptorfamily members are reported. Experimental GPCR profiling indicated thatONC201 selectively antagonizes the D2-like, but not D1-like, dopaminereceptor subfamily Reporter assays in a heterologous expression systemrevealed that ONC201 selectively antagonizes both short and longisoforms of DRD2 and DRD3, with weaker potency for DRD4 and noantagonism of DRD1 or DRD5. Increased secretion of prolactin is aclinical hallmark of DRD2 antagonism by several psychiatric medicationsthat potently target this receptor. ELISA measurements in peripheralblood of patients treated with ONC201 in the first-in-human trial withadvanced solid tumors determined that 10/11 patients evaluated exhibitedinduction of prolactin (mean of 2-fold).

Using the TCGA database, the D2-like dopamine receptor subfamily,particularly DRD2, was found to be prevalent and selectivelyoverexpressed in several malignancies. Preclinical reports show thatDRD2 inhibition imparts antitumor efficacy, without killing normalcells, via induction of ATF4/CHOP and inhibition of Akt and ERKsignaling that are all attributes of ONC201.

Methods

ONC201 dihydrochloride was obtained from Oncoceutics. Kinase inhibitionassays for the kinome were performed as described (see Anastassiadis etal., Nat Biotech 29:1039 (2011)). GPCR arrestin recruitment and cAMPmodulation reporter assays were performed as described (see McGuinnesset al., Journal of Biomolecular Screening 14:49 (2009)). PathHunter™(DiscoveRx) β-arrestin cells expressing one of several GPCR targets wereplated onto 384-well white solid bottom assay plates (Corning 3570) at5000 cells per well in a 20 μL volume in an appropriate cell platingreagent. Cells were incubated at 37° C., 5% CO₂ for 18-24 h. Sampleswere prepared in buffer containing 0.05% fatty-acid free BSA (Sigma).For agonist mode tests, samples (5 μL) were added to pre-plated cellsand incubated for 90 minutes at 37° C., 5% CO₂. For antagonist modetests, samples (5 μL) were added to pre-plated cells and incubated for30 minutes at 37° C., 5% CO₂ followed by addition of EC₈₀ agonist (5 μL)for 90 minutes at 37° C., 5% CO₂. For Schild analysis, samples (5 μL)were added to pre-plated cells and incubated for 30 minutes at 37° C.,5% CO₂ followed by addition of serially diluted agonist (5 μL) for 90minutes at 37° C., 5% CO₂. Control wells defining the maximal andminimal response for each assay mode were tested in parallel. Arrestinrecruitment was measured by addition of 15 μL PathHunter Detectionreagent and incubated for 1-2 h at room temperature and read on a PerkinElmer Envision Plate Reader. For agonist and antagonist tests, data wasnormalized for percent efficacy using the appropriate controls andfitted to a sigmoidal dose-response (variable slope),Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((LogEC₅₀−X)*HillSlope)), where X is the log concentration of compound. ForSchild analysis, data was normalized for percent efficacy using theappropriate controls and fitted to a Gaddum/Schild EC₅₀ shift usingglobal fitting, where Y=Bottom+(Top−Bottom)/(1+10{circumflex over( )}((Log EC−X)*HillSlope)), Antag=1+(B/(10{circumflex over( )}(−1*pA2))){circumflex over ( )}SchildSlope and LogEC=Log(EC₅₀*Antag). EC₅₀/IC₅₀ analysis was performed in CBIS dataanalysis suite (Cheminnovation) and Schild analysis performed inGraphPad Prism 6.0.5.

Results

ONC201 is a small molecule in phase II clinical trials for selectadvanced cancers. It was discovered in a phenotypic screen forp53-independent inducers of the pro-apoptotic TRAIL pathway. Althoughthe contribution of ONC201-induced ATF4/CHOP upregulation andinactivation of Akt/ERK signaling (Allen et al., Science translationalmedicine 5, 171ra117-171ra117 (2013)) to its anti-cancer activity hasbeen characterized, its molecular binding target has remained elusive.

In vitro profiling of GPCR activity using a heterologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC201 selectively antagonizes the D2-like (DRD2/3/4), but not D1-like(DRD1/5), dopamine receptor subfamily (FIGS. 4B and 5A). Antagonism ofadrenoceptor alpha receptors or other GPCRs was not observed under theevaluated conditions. Among the DRD2 family, ONC201 antagonized bothshort and long isoforms of DRD2 and DRD3, with weaker potency for DRD4.Further characterization of ONC201-mediated antagonism of arrestinrecruitment to DRD2L was assessed by a Gaddum/Schild EC₅₀ shiftanalysis, which determined a dissociation constant of 2.9 μM for ONC201that is equivalent to its effective dose in many human cancer cells(FIG. 4C). Confirmatory results were obtained for cAMP modulation inresponse to ONC201, which is another measure of DRD2L activation (FIG.4D). The ability of dopamine to reverse the dose-dependent antagonism ofup to 100 μM ONC201 suggests direct, competitive antagonism of DRD2L(FIGS. 5B and 5C). In agreement with the ONC201 specificity predicted byBANDIT, no significant interactions were identified between ONC201 andnuclear hormone receptors, the kinome, or other drug targets ofFDA-approved cancer therapies (FIGS. 5D and 5E). Interestingly, abiologically inactive constitutional isomer of ONC201 (Wagner et al.,Oncotarget 5:12728 (2014)) did not inhibit DRD2L, suggesting thatantagonism of this receptor could be linked to its biological activity(FIG. 5F). In summary, these studies establish that ONC201 selectivelyantagonizes the D2-like dopamine receptor subfamily, which appears to bea promising therapeutic target in oncology, and ONC201 is the firstcompound to exploit this treatment paradigm in several ongoing Phase IIclinical studies.

Example 13: Shotgun Mutagenesis Epitope Mapping of DRD2

Shotgun Mutagenesis uses a high-throughput cellular expressiontechnology to express and analyze large libraries of mutated targetproteins within eukaryotic cells. Every residue in a protein isindividually mutated to an alanine, or other specified residue, to assaychanges in function. Proteins are expressed within standard mammaliancell lines, therefore even difficult proteins that require eukaryotictranslational or post-translational processing can be mapped.

First, conditions were evaluate and identified for screening the DRD2antagonist ONC201 with wild-type DRD2 using the Shotgun Mutagenesisscreening assay. Then, a DRD2 Ala-scan library was prepared and theresidues critical for ONC201 binding were mapped at single amino acidresolution using Shotgun Mutagenesis technology.

DRD2 Shotgun Mutagenesis Library:

Parental plasmid: DRD2

Library size: 442 mutant clones (complete protein)

Mutation Strategy: Alanine scan mutagenesis

Cell type: HEK-293T

Screening Assay: Calcium flux

Epitope Tag: C-terminal V5/HIS6

Parental Construct: DNA encoding the full-length human DRD2 (AccessionNo: NP_000786.1; MDPLNLSWYD DDLERQNWSR PFNGSDGKAD RPHYNYYATL LTLLIAVIVFGNVLVCMAVS REKALQTTTN YLIVSLAVAD LLVATLVMPW VVYLEVVGEW KFSRIHCDIFVTLDVMMCTA SILNLCAISI DRYTAVAMPM LYNTRYSSKR RVTVMISIVW VLSFTISCPLLFGLNNADQN ECIIANPAFV VYSSIVSFYV PFIVTLLVYI KIYIVLRRRR KRVNTKRSSRAFRAHLRAPL KGNCTHPEDM KLCTVIMKSN GSFPVNRRRV EAARRAQELE MEMLSSTSPPERTRYSPIPP SHHQLTLPDP SHHGLHSTPD SPAKPEKNGH AKDHPKIAKI FEIQTMPNGKTRTSLKTMSR RKLSQQKEKK ATQMLAIVLG VFIICWLPFF ITHILNIHCD CNIPPVLYSAFTWLGYVNSA VNPIIYTTFN IEFRKAFLKI LHC (SEQ ID NO: 1) was subcloned into amammalian high-expression vector. This parental construct wassequence-verified and then validated for mammalian cell expression bydetection of calcium flux in response to dopamine DNA yields fromplasmid preparations have been validated for high-throughput processing.

Assay Set-up: A DRD2-specific calcium flux assay was successfullyoptimized for DRD2 expressed in human cells. An agonist dose-responseassay was used to identify a suitable dopamine concentration for use inoptimizing the inhibition of DRD2-specific calcium flux by antagonistONC201. Subsequent dose-response inhibition assays identified aconcentration of ONC201 that inhibited the DRD2 dopamine response by>95%.

Calcium Flux Assay Optimization:

Receptor Activity Assay.

DRD2 activity was assessed using a published GPCR assay (Greene, T. A.et al., (2011) PLoS One 6, e20123). Briefly, HEK-293T cells weretransfected with expression constructs for wild-type DRD2 or a negativecontrol GPCR, in 384-well format. After 22 hr, calcium flux experimentswere performed over a range of dopamine concentrations (300 pM-100 nM),using a Flexstation II-384 fluorescence reader (Molecular Devices). Datasets were analyzed and represented as percentage over baseline signalusing Prism 5.0 software (GraphPad Software, Inc).

For cells expressing DRD2, but not a control GPCR, addition of dopamineresulted in increases in cellular calcium flux, measured as increasedfluorescence. A dose response plot of the fluorescence peak heightversus dopamine concentration demonstrated the strong dopamine-inducedcalcium flux (EC₅₀=0.45 nM) in cells expressing DRD2, but not thecontrol GPCR. This suggested that the calcium flux assay could be usedto test for ONC201 inhibition.

DRD2 Calcium Flux Inhibition Assay Optimization

Following identification of the EC₅₀ for dopamine in the calcium fluxassay, ONC201 inhibition of DRD2-specific calcium flux was investigatedat several dopamine concentrations. Using 1 nM dopamine (>2-fold higherthan the dopamine EC₅₀) with a range of ONC201 concentrations (1 nM to100 μM), ONC201 inhibition of dopamine-induced DRD2 calcium flux wasobserved at the highest concentrations tested (FIG. 9A), with completeinhibition by 100 μM ONC201 (IC₅₀=21.5 μM). Inhibition of calcium fluxby 100 μM ONC201 was not the result of a broad inhibition of GPCRs or ofa non-specific effect on cells since ONC201 had no effect on the calciumflux activity of cells expressing a control GPCR (FIG. 9B).

Analysis of a number of replicate values obtained for inhibition of DRD2calcium flux by 100 μM ONC201 indicated a robust assay, with a Z′ valueof 0.61. The Z′ value is a measurement of assay quality, calculated fromthe means and standard deviations obtained for replicate determinationsof calcium flux obtained with or without ONC201.

Comparison of DRD2 Inhibitors.

The ONC201 inhibition of DRD2 was compared to that by the DRD2antagonists spiperone and domperidone (FIG. 10), which have beendescribed as inhibiting DRD2 at concentrations lower than the 100 μMrequired for inhibition by ONC201. These antagonists were screened atconcentrations between 100 μM and 1 μM, and both showed completeinhibition of dopamine-induced calcium flux, with spiperone having anIC₅₀=19 nM, and domperidone an IC₅₀=47 nM. These values were consistentwith previous characterizations and demonstrate that the relatively highIC₅₀ obtained for ONC201 (21.5 μM) does not result from the use of acalcium flux assay to measure DRD2 activity.

Optimal screening conditions were determined for ONC201 inhibition ofDRD2-specific calcium flux in response to dopamine. These conditionsgive a robust response to dopamine, this response is reduced by >95% byaddition of ONC201 to 100 μM, and the assay demonstrated low variabilitybetween replicates. These data indicate that the selected conditions aresuitable for successful high-throughput screening. Further screening ofthe DRD2 mutation library was at a dopamine concentration of 1 nM and anONC201 concentration of 100 μM.

Screening the DRD2 Alanine-Scan Library for Response to Dopamine.

The DRD2 alanine-scan mutation library (and with alanines changed toserines) comprised 442 clones, covering residues 2-443 of the DRD2protein, 100% of target residues. The DRD2 mutation library was firstscreened by calcium flux assay with dopamine (1 nM) in the absence ofONC201 to identify residues whose mutation diminished dopamine-inducedcalcium flux. We identified 28 amino acid residues that were criticalfor dopamine-induced DRD2 flux (FIG. 11).

Residues were identified from the analysis are listed in Table 4 andshown in FIG. 11. Clones were considered to be deficient for calciumflux if they demonstrated flux values less than 2 standard deviationsbelow the average calcium flux value (AV−2SD) for the entire library.

TABLE 4 DRD2 RESIDUES CRITICAL FOR DOPAMINE-INDUCED CALCIUM FLUX CalciumFlux Calcium Flux Mutation % WT Mutation % WT C182A 0 S7A 15 I184A 0W386A 15 S197A 0 S121A 16 T119A 1 I394A 16 S193A 1 E248A 19 D80A 3 V190A20 R132A 3 Y199A 20 D114A 4 C107A 20 H393A 4 S419A 20 F198A 10 F189A 22V83A 10 I122A 23 I377A 11 T205A 24 Y416A 12 N23A 25 C118A 14 L125A 25I128A 27Screening the DRD2 Alanine-Scan Library for ONC201 Inhibition ofDopamine-Induced Signaling Identified Residues Required for Inhibitionby ONC201.

To identify residues important for the inhibition of DRD2 by ONC201, theDRD2 alanine-scan mutation library was screened by the calcium fluxassay for the ability to respond to dopamine in the presence of aninhibiting concentration of ONC201, using dopamine at 1 nM and ONC201 at100 μM. Eight residues critical for ONC201 inhibitory activity wereidentified (FIG. 12). All residues identified by this screen showed highcalcium flux with dopamine alone (Table 5). Clones were considered to becritical for inhibition by ONC201 at 100 μM if they demonstrated fluxvalues greater than 2 standard deviations above the average calcium fluxvalue (AV+2SD) for the entire library. Also shown in Table 5 for thesecritical clones are the calcium flux values obtained from similarexperiments performed with 250 μM ONC201 or without ONC201 (dopamine 1nM), and in addition the % conservation of the critical residues acrossthe 5 DRD receptors, with the residues found in each receptor.

TABLE 5 DRD2 RESIDUES CRITICAL FOR ONC201 INHIBITION OF DOPAMINE-INDUCEDCALCIUM FLUX Calcium Flux as a % of flux with WT DRD2 (100) DRD % DRDMutation ONC201 100 μM ONC201 250 μM Dopamine 1 nM Conservation 1 2 3 45 I397A 122 89 105 20 P I T A P E95A 97 39 123 100 E E E E E V91A 94 58119 40 K V V F K Y192A 85 11 64 60 S Y Y Y S V196A 79 22 119 40 I V V CI A177S 77 26 85 40 A A T V D T165A 67 28 92 20 L T A A L L81A 63 20 83100 L L L L L

Since the average inhibition by 100 μM ONC201 across the library wasapproximately 75%, we also conducted a screen at 250 μM ONC201 todetermine if critical residues would be the same at higher levels ofinhibition. Under this condition dopamine-induced calcium flux wasinhibited by approximately 93%, and the previously identified residuesV91, E95, and 1397 were also critical for inhibition at 250 μM ONC201(Table 5), using the same criteria of flux values greater than 2standard deviations above the average calcium flux value (AV+2SD) forthe library.

Conclusions:

In initial screens of the DRD2 alanine-scan mutation library bydopamine-induced calcium flux assay, 28 mutations greatly decreasedcalcium flux, identifying resides critical for DRD2 function. As foundin a similar analysis of the GPCR CXCR4, the critical residues weredistributed throughout the protein, in the predicted dopamine bindingpocket, the transmembrane regions and in the cytoplasmic exposed portionof DRD2. These 28 residues are critical for either dopamine binding,signal transduction through the transmembrane domains, or G proteincoupling. A detailed analysis comparable to that performed for CXCR4, aswell as the structural analysis of the DRD3-eticlopride structure (Chienet al., 2010), can be used to assign specific function to each DRD2critical residue.

To identify residues important for the inhibition of DRD2 by ONC201, theDRD2 alanine-scan mutation library was screened by calcium flux assaywith dopamine and 100 μM ONC201. These screens identified 8 residues ascritical for ONC201 inhibition of DRD2-dependent dopamine-inducedcalcium flux—L81, V91, E95, T165, A177, Y192, V196, and I397. ResiduesV91, E95, and I397 were also identified as critical for resistance toDRD2 inhibition by 250 μM ONC201, suggesting that they are keyONC201-interacting residues. These residues define a relatively largeligand binding site, which is not unexpected due to the larger size ofONC201 compared to dopamine and eticlopride. The locations of theseresidues are generally consistent with a role in mediating ONC201inhibition of DRD2-dependent dopamine-induced calcium flux. Residuescritical for inhibition of a GPCR taste receptor by probenecid werepreviously identified (Greene et al., 2011), with the location of theresidues consistent with a non-competitive mechanism of inhibition. Incontrast, the residues identified here for DRD2 are consistent withcompetitive inhibition by ONC201 at the dopamine binding site. Whenmodeled on the structure of homologous receptor DRD3, the majority ofthe residues identified surround the binding pocket containing aco-crystallized antagonist eticlopride, with 5 of the 8 identifiedresidues conserved between DRD2 and DRD3. Two of the residues appear tomore distal from the putative binding site (A177 and L81) and may affectONC201 binding in a more allosteric fashion. Additional residues thatcontribute to ONC201 inhibition may be identified using DRD2 agonistswith structures distinct from dopamine.

Example 14: Determination of the Association & Dissociation RateConstants of Unlabelled ONC201 Dihydrochloride on the Human D2S Receptor

In this Example, the kon/koff rates of unlabeled ONC201 dihydrochlorideon the D2S receptor was determined. The kon/koff rate estimation wasperformed by competitive ligand binding according to the methoddescribed in: M. R. Dowling & S. J. Charlton (2006) Brit. J. Pharmacol.148:927-937 and H. J. Motulsky & L. C. Mahan (1984) Mol. Pharmacol.25:1-9. Referring to this method, the kon/koff rates of the unlabeledtest compounds were calculated from its Ki value (competition binding)and its effect on the binding kinetics of the radioligand (competitionkinetics).

First, the IC₅₀ and Ki values of ONC201 dihydrochloride, and selectionof the adequate compound concentrations for the competition kineticsexperiment, were determined. Then, the kon and koff rate constants ofthe radioligand ([³H]Methylspiperone) was determined Finally, the konand koff rate constants of the unlabeled ONC201 dihydrochloride wasdetermined. ONC201 dihydrochloride was tested at 8 concentrations induplicate (n=2) in the competition binding assay, and the IC₅₀ and Kivalues were determined.

The reference compound, (+) Butaclamol, and the test compound,ONC201-2HCL, successfully competed for [³H]Methylspiperone, with IC₅₀values of 2.5 nM and 21 μM, respectively. Previously, the compoundONC201-2HCL yielded a similar IC₅₀ value of 16 μM. For the competitionbinding assay, the following 6 concentrations of ONC201-2HCL wereselected: 5/10/20/40/60/80 μM.

The binding kinetics of [³H]Methylspiperone on the D2S receptor wasdetermined. For this, [³H]Methylspiperone (at one concentration of 0.3nM) was incubated with the D2S receptor membranes for 12 differentincubation times to measure the association rate. The non-specificbinding was measured with Butaclamol (10 μM) for each incubation time.The dissociation was initiated by addition of an excess of Butaclamol(10 μM) after 60 minutes incubation of [³H]Methylspiperone (0.3 nM) withthe D2S receptor membranes, and the signal decrease was measured after12 different incubation times. The experiment was performed intriplicate (n=3) with incubation times adjusted to0/30/60/80/120/180/240/300/360/420/480 minutes and 24 hours for theassociation and 2/5/8/10/15/20/25/30/40/60/120/180 minutes for thedissociation kinetics.

[3H]Methylspiperone displayed a k_(on) value of 2.3×10⁸ M⁻¹min⁻¹ and ak_(off) value of 0.009506 min⁻¹ (and thus a t_(1/2) value of 73 minutes)on the D2S receptor. The K_(d) calculated from the results of theassociation/dissociation experiment (0.04 nM) is in the same range ascompared to the K_(d) observed in the saturation experiment (0.15 nM),thereby validating the experiment.

The effect of the unlabeled ONC201-2HCl at six concentrations on theassociation kinetics of [³H]Methylspiperone (0.3 nM) was tested. Thenon-specific binding was measured with Butaclamol (10 μM). The same 12incubation times as above were used: 2/5/8/10/15/20/25/30/40/60/120/180minutes. A measurement in the absence of compound was performed asnegative control.

ONC201-2HCl displayed a k_(on) value of 4.1×10⁵ M⁻¹min⁻¹ and a k_(off)value of 1.32 min⁻¹ (and thus a t_(1/2) value of 0.53 minutes) on theD2S receptor. The K_(i) calculated from the results of theassociation/dissociation experiment (3.2 μM) is in the same range ascompared to the K_(i) observed in the saturation experiment (7 μM),thereby validating the experiment. In conclusion, ONC201-2HCl displays amuch slower association and a much faster dissociation as compared to[³H]Methylspiperone.

Example 15: Bactericidal Activity of Imipridones

Materials and Methods

Test material: ONC201 dihydrochloride; Control: MicrocrystallineCellulose.

Method: Harmonized EP/USP Microbial Examination of Nonsterile Products(Current USP <61>/<62>).

Results

TABLE 6 VERIFICATION OF THE INOCULUM RECOVERY CONTROL AND MICROBIALENUMERATION TEST 1:300 with TSB Indicator Organisms Count Mod DilutionEc Sa Pa Bs Ca (TSA) Ab (TSA) CA (SDA) Ab (SDA) Inoculum 27 31 28 52 4821 52 20 434019 N/A 0 24 48 51 18 46 19

TABLE 7 THE VALIDATION FOR SPECIFIED MICROORGANISMS Sample BTGN Ec Pa SaCa 1:300 with P P P F P TSB Mod Dilution P = Pass F = Fail NA = NotApplicable; Ec = Escherichia coli ATCC# 8739; Pa = Pseudomonasaeruginosa ATCC# 9027; Sa = Staphylococcus aureus ATCC# 6538; Bs =Bacillus subtilis ATCC# 6633; Ca = Candida albicans ATCC# 10231; Ab =Aspergillus brasiliensis ATCC# 16404; BTGN = Bile Tolerant Gram Negativebacteria; Cs = Clostridium species; TSA = Trypticase Soy Agar; SDA =Sabouraud Dextrose Agar.

ONC201 dihydrochloride when tested at the 1:300 dilution with TSB Mod,did not meet the requirements of the USP <61>/<62>

Microbial Limit Suitability Test. Inhibition was observed forStaphylococcus aureus for USP<61>/<62>. Therefore, it can be assumedthat the failure to isolate the inoculated microorganism is attributableto the bactericidal activity of ONC201 dihydrochloride and thus it isnot likely to be contaminated with the inhibited species ofmicroorganism.

Next, the Minimal Inhibitory Concentration (MIC) for six imipridones wasdetermined against wild type and methicillin-resistant Staphylococcusaureus.

Materials and Methods

Compounds

ONC201 and ONC206 were previously solubilized at 40 mM in DMSO. ONC212,ONC207 and ONC213 were solubilized at 20 mg/mL in DMSO and an ONC201linear isomer (TIC-10) was solubilized at 10 mg/mL in DMSO. Methicillinand/or vancomycin were evaluated in parallel as positive controlantibiotics and were purchased from Sigma-Aldrich and solubilized indeionized H₂O at a concentration of 10 mg/mL.

Bacteria

The bacterial strains employed in these assays were obtained from theAmerican Type Culture Collection (ATCC). All bacterial strains werepropagated as recommended by the ATCC. Each strain was stored as afrozen glycerol stock at −80° C. and a 10 μL loop of the frozen stockwas used to inoculate each culture for these assays. The strains withtheir classification and properties are listed in Table 8 below.

TABLE 8 STRAINS OF STAPHYLOCOCCUS AUREUS AND CHARACTERISTICS ATCC #Classification Properties Assay Media 29213 Gram QC Wild Trypticase SoyPositive Type Strain Broth (TSB) 33591 Cocci Hospital Acquired NutrientBroth Methicillin Resistant 700699 Hospital Brain Heart Acquired, MDR,Infusion Broth + Reduced 0.004 g/L Susceptibility Vancomycin toVancomycinMinimal Inhibitory Concentration (MIC) Determination

The susceptibility of the bacterial organisms to the test compounds wasevaluated by determining the MIC of each compound using a micro-brothdilution analysis according to the methods recommended by the Clinicaland Laboratory Standards Institute CLSI. All microbial strains wereobtained from American Type Culture Collections (ATCC) and culturedaccording to the supplier's recommendations. Evaluation of thesusceptibility of each organism against the test compounds included apositive control antibiotic(s). For each organism, a standardizedinoculum was prepared by direct suspension of freshly plated colonies inthe appropriate media as indicated in Table 8 to an optical density 625nm (OD₆₂₅) of 0.1 (equivalent to a 0.5 McFarland standard). Thesuspended inoculum was diluted to a concentration of approximately 1×10⁶colony forming units per milliliter (CFU/mL) and 100 μL placed intotriplicate wells of a 96-well plate containing 100 μL of test compoundserially diluted 2-fold in the appropriate broth. One hundredmicroliters (100 μL) of the inoculum was also added to triplicate wellscontaining 100 μL of two-fold serial dilutions of a positive controlantibiotic and to wells containing 100 μL of media only. This dilutionscheme yielded final concentrations for each microbial organismestimated to be 5×10⁵ CFU/mL. Test compound concentrations ranged from ahigh-test of 100 to a low test of 0.2 μM using a two-fold dilutionscheme. The plates were incubated for 24 or 48 hours (Staphylococcusaureus 700699) at 37° C. and the microbial growth at each concentrationof compound was determined by measuring the optical density at 625 nm ona Molecular Devices SpectraMax Plus-384 plate reader and visually byscoring the plates+/− for bacterial growth. The MIC for each compoundwas determined as the lowest compound dilution that completely inhibitedmicrobial growth.

Results

Six (6) imipridones were evaluated for their ability to inhibit thegrowth of three strains of Staphylococcus aureus. ONC201, ONC207, and anONC201 linear isomer (TIC-10) were inactive against all three strains upto a concentration of 100 μg/mL. Against wild type Staphylococcus aureus(ATCC 29213) the MIC of ONC206, ONC212 and ONC213 was 6.25 μg/mL, 3.13μg/mL and 25 μg/mL, respectively. Against Staphylococcus aureus (ATCC33591) the MIC of ONC206, ONC212 and ONC213 was 12.5 μg/mL, 3.13 μg/mLand 3.13 μg/mL, respectively. The activity was similar against the MDRStaphylococcus aureus (ATCC 700699) with all three compounds having aMIC of 12.5 μg/mL. Vancomycin, the positive control compound, was activeat the expected concentration and methicillin was found to be inactiveup to a concentration of 100 μg/mL against the two methicillin resistantstrains of bacteria. Data are presented in Table 9.

TABLE 9 MIC DETERMINATION OF 6 IMIPRIDONES FOR 3 STAPHYLOCOCCUS AUREUSSTRAINS Staphylococcus aureus Staphylococcus aureus Staphylococcusaureus Compound ATCC 29213 ATCC 33591 ATCC 700699 (48 hours) (μg/mL)MIC₉₀ MIC₉₅ MIC₉₉ Visual MIC₉₀ MIC₉₅ MIC₉₉ Visual MIC₉₀ MIC₉₅ MIC₉₉VisualONC201 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100ONC206 6.25 6.25 6.25 6.25 12.5 25 >100 12.5 12.5 12.5 25 12.5ONC207 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100ONC212 3.13 3.13 3.13 3.125 3.13 6.25 100 3.125 6.25 12.5 12.5 12.5ONC213 12.5 12.5 25 25 3.13 6.25 100 3.125 6.25 12.5 12.5 12.5TIC-10 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100Methicillin — — — — >100 >100 >100 >100 >100 >100 >100 >100 Vancomycin3.13 3.13 6.25 3.125 0.39 0.39 0.78 0.391 12.5 25 25 25Discussion

Six (6) imipridones were evaluated for activity against 3 strains ofStaphylococcus aureus. ONC201, ONC207, and TIC-10 were inactive againstall three strains. ONC206, ONC212 and ONC213 had varying activityranging from 3.13 μg/mL to 25 μg/mL against all three bacterial strains.Relative to vancomycin the activity of these three imipridones wasequivalent or 2 to 8-fold less against strain 29213. All three of theseimipridones had 10 to 30-fold less activity compared to vancomycinagainst strain 33591 and the activity for all three compounds was 2-foldhigher than vancomycin against strain 700699.

These experiments are repeated with additional impiridones and foradditional bacteria, including both Gram-positive and Gram-negativebacteria, such as those in Table 10.

TABLE 10 Gram +/ Organism Condition Gram− Enterococcus Noscomialbacteremia, wound + faecium infections, endocarditis, UTIsStaphylococcus Bacteremia, endocarditis + aureus Klebsiella Pneumonia,UTIs, Upper − pneumonia respiratory tract infections AcinetobacterInfections in ICU and − baumannii burn patients; also being seen ingeneral hospital and nursing homes Pseudomonas Pneumoniae, CF −aeruginosa Enterobacter UTIs, respiratory infections − cloacae

Example 16: Case Study of ONC201 Treatment in a Subject with RecurrentGlioblastoma

This Example provides a case study of a 22 year old female withrecurrent glioblastoma (unmethylated MGMT, H3.3 K27M mutant) treatedwith 625 mg of ONC201 once every three weeks. FIG. 28 (A) Tumor sizerelative to baseline (%) of total tumor burden in the subject. One cycleis 3 weeks. (B) Contrast MRI scans at baseline, 21, 27 and 36 weekspost-ONC201 initiation of one of 2 malignant lesions in the subject with625 mg q3w ONC201.

It will be appreciated by one skilled in the art that changes could bemade to the exemplary embodiments shown and described above withoutdeparting from the broad inventive concept thereof. It is understood,therefore, that this invention is not limited to the exemplaryembodiments shown and described, but it is intended to covermodifications within the spirit and scope of this invention as definedby the claims. For example, specific features of the exemplaryembodiments may or may not be part of the claimed invention and featuresof the disclosed embodiments may be combined. Unless specifically setforth here, the terms “a”, “an” and “the” are not limited to one elementbut instead should be read to mean “at least one.”

It is to be understood that the figures and descriptions may have beensimplified to focus on elements that are relevant for a clearunderstanding, while eliminating, for purposes of clarity, otherelements that those of ordinary skill in the art will appreciate mayalso comprise a portion of the invention. However, because such elementsare well known in the art, and because they do not necessarilyfacilitate a better understanding of the invention, a description ofsuch elements is not provided herein.

Further, to the extent that a method does not rely on the particularorder of steps set forth, the particular order should not be construedas limitation on the claims. Claims directed to a method should not belimited to performance of the steps in the order written, and oneskilled in the art can readily appreciate that they can be varied andstill remain within the spirit and scope of this invention.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entirety here.

What is claimed is:
 1. A method of treating cancer in a subject in needthereof, comprising: administering to the subject in need of suchtreatment a pharmaceutical composition comprising a therapeuticallyeffective amount of compound (1)

or a pharmaceutically acceptable salt thereof, wherein the cancer is aneuroendocrine cancer, and wherein the neuroendocrine cancer is selectedfrom a pheochromocytoma or a paraganglioma.
 2. The method according toclaim 1, wherein the cancer has a histone H3 mutation.
 3. The methodaccording to claim 2, wherein the histone H3 mutation is H3.3 K27M. 4.The method according to claim 1, wherein the cancer has anepigenetically silenced unmethylated O(6)-methylguanine-DNAmethyltransferase (MGMT) gene.